Broad neutralization of SARS-CoV-2 variants by an inhalable bispecific single-domain antibody.

The effectiveness of SARS-CoV-2 vaccines and therapeutic antibodies have been limited by the continuous emergence of viral variants and by the restricted diffusion of antibodies from circulation into the sites of respiratory virus infection. Here, we report the identification of two highly conserved regions on the Omicron variant receptor-binding domain recognized by broadly neutralizing antibodies. Furthermore, we generated a bispecific single-domain antibody that was able to simultaneously and synergistically bind these two regions on a single Omicron variant receptor-binding domain as revealed by cryo-EM structures. We demonstrated that this bispecific antibody can be effectively delivered to lung via inhalation administration and exhibits exquisite neutralization breadth and therapeutic efficacy in mouse models of SARS-CoV-2 infections. Importantly, this study also deciphered an uncommon and highly conserved cryptic epitope within the spike trimeric interface that may have implications for the design of broadly protective SARS-CoV-2 vaccines and therapeutics.

Antivirals with common targets against highly pathogenic viruses.

Historically, emerging viruses appear constantly and have cost millions of human lives. Currently, climate change and intense globalization have created favorable conditions for viral transmission. Therefore, effective antivirals, especially those targeting the conserved protein in multiple unrelated viruses, such as the compounds targeting RNA-dependent RNA polymerase, are urgently needed to combat more emerging and re-emerging viruses in the future. Here we reviewed the development of antivirals with common targets, including those against the same protein across viruses, or the same viral function, to provide clues for development of antivirals for future epidemics.

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

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

SARS-CoV-2 evolution from the BA.2.86 to JN.1 variants: unexpected consequences.

SARS-CoV-2 is continuously evolving. The Omicron subvariant BA.2.86, with >30 mutations in its spike (S) protein compared with its predecessor strain BA.2, was expected to quickly become predominant worldwide, but this has not happened. Instead, its descendant strain, JN.1, with just one additional mutation, has become the predominant SARS-CoV-2 subvariant. Here, we offer a possible explanation for these unexpected consequences.

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

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

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

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

Human airway and nasal organoids reveal escalating replicative fitness of SARS-CoV-2 emerging variants.

The high transmissibility of SARS-CoV-2 Omicron subvariants was generally ascribed to immune escape. It remained unclear whether the emerging variants have gradually acquired replicative fitness in human respiratory epithelial cells. We sought to evaluate the replicative fitness of BA.5 and earlier variants in physiologically active respiratory organoids. BA.5 exhibited a dramatically increased replicative capacity and infectivity than B.1.1.529 and an ancestral strain wildtype (WT) in human nasal and airway organoids. BA.5 spike pseudovirus showed a significantly higher entry efficiency than that carrying WT or B.1.1.529 spike. Notably, we observed prominent syncytium formation in BA.5-infected nasal and airway organoids, albeit elusive in WT- and B.1.1.529-infected organoids. BA.5 spike-triggered syncytium formation was verified by lentiviral overexpression of spike in nasal organoids. Moreover, BA.5 replicated modestly in alveolar organoids, with a significantly lower titer than B.1.1.529 and WT. Collectively, the higher entry efficiency and fusogenic activity of BA.5 spike potentiated viral spread through syncytium formation in the human airway epithelium, leading to enhanced replicative fitness and immune evasion, whereas the attenuated replicative capacity of BA.5 in the alveolar organoids may account for its benign clinical manifestation.

Developing pan-ß-coronavirus vaccines against emerging SARS-CoV-2 variants of concern.

The concurrent prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV) raises the concern for the emergence of potential new ß-CoV clades via genetic recombination, bearing high SARS-CoV-2-like transmissibility and high MERS-CoV-like mortality rates. Therefore, we argue that there is an urgent need to develop pan-ß-CoV vaccines that can target not only current SARS-CoV-2 variants of concern, but also future putative SARS-CoV-3- or MERS-CoV-2-like coronavirus.

A subunit-based influenza/SARS-CoV-2 Omicron combined vaccine induced potent protective immunity in BALB/c mice.

Infection with influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a significant risk to human life, health, and the global economy. Vaccination is one of the most effective strategies in the fight against infectious viruses. In this study, we, for the first time, have evaluated the immunogenicity and protective effect of an influenza/SARS-CoV-2 Omicron subunit combined vaccine adjuvanted with MF59 and administered to BALB/c mice. Results showed that the combined vaccine induced high levels of IgG, IgG1 , and IgG2a antibodies, as well as influenza A H1N1/California/2009 virus-specific hemagglutination-inhibiting antibodies in BALB/c mice. Moreover, this subunit combined vaccine induced high titers of neutralization antibodies against SARS-CoV-2 Omicron sublineage BA.5 pseudovirus and effectively reduced the viral load of authentic SARS-CoV-2 Omicron sublineage BA.5.2 in the cell culture supernatants. These results suggested that this subunit combined vaccine achieved protective effect against both H1N1 A/California/07/2009 strain and SARS-CoV-2 Omicron BA.5.2 variant. It is therefore expected that this study will establish the scientific foundation for the next-step development of combined vaccines against other strains or variants of IAV and SARS-CoV-2.

Design of MERS-CoV entry inhibitory short peptides based on helix-stabilizing strategies.

Interaction between Middle East respiratory syndrome coronavirus (MERS-CoV) spike (S) protein heptad repeat-1 domain (HR1) and heptad repeat-2 domain (HR2) is critical for the MERS-CoV fusion process. This interaction is mediated by the α-helical region from HR2 and the hydrophobic groove in a central HR1 trimeric coiled coil. We sought to develop a short peptidomimetic to act as a MERS-CoV fusion inhibitor by reproducing the key recognition features of HR2 helix. This was achieved by the use of helix-stabilizing strategies, including substitution with unnatural helix-favoring amino acids, introduction of ion pair interactions, and conjugation of palmitic acid. The resulting 23-mer lipopeptide, termed AEEA-C16, inhibits MERS-CoV S protein-mediated cell-cell fusion at a low micromolar level comparable to that of the 36-mer HR2 peptide HR2P-M2. Collectively, our studies provide new insights into developing short peptide-based antiviral agents to treat MERS-CoV infection.

Cross-reactive antibody response to Monkeypox virus surface proteins in a small proportion of individuals with and without Chinese smallpox vaccination history.

BACKGROUND: After the eradication of smallpox in China in 1979, vaccination with the vaccinia virus (VACV) Tiantan strain for the general population was stopped in 1980. As the monkeypox virus (MPXV) is rapidly spreading in the world, we would like to investigate whether the individuals with historic VACV Tiantan strain vaccination, even after more than 40 years, could still provide ELISA reactivity and neutralizing protection; and whether the unvaccinated individuals have no antibody reactivity against MPXV at all. RESULTS: We established serologic ELISA to measure the serum anti-MPXV titer by using immunodominant MPXV surface proteins, A35R, B6R, A29L, and M1R. A small proportion of individuals (born before 1980) with historic VACV Tiantan strain vaccination exhibited serum ELISA cross-reactivity against these MPXV surface proteins. Consistently, these donors also showed ELISA seropositivity and serum neutralization against VACV Tiantan strain. However, surprisingly, some unvaccinated young adults (born after 1980) also showed potent serum ELISA activity against MPXV proteins, possibly due to their past infection by some self-limiting Orthopoxvirus (OPXV). CONCLUSIONS: We report the serum ELISA cross-reactivity against MPXV surface protein in a small proportion of individuals both with and without VACV Tiantan strain vaccination history. Combined with our serum neutralization assay against VACV and the recent literature about mice vaccinated with VACV Tiantan strain, our study confirmed the anti-MPXV cross-reactivity and cross-neutralization of smallpox vaccine using VACV Tiantan strain. Therefore, it is necessary to restart the smallpox vaccination program in high risk populations.

Sensitivity to Vaccines, Therapeutic Antibodies, and Viral Entry Inhibitors and Advances To Counter the SARS-CoV-2 Omicron Variant.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) keeps evolving and mutating into newer variants over time, which gain higher transmissibility, disease severity, and spread in communities at a faster rate, resulting in multiple waves of surge in Coronavirus Disease 2019 (COVID-19) cases. A highly mutated and transmissible SARS-CoV-2 Omicron variant has recently emerged, driving the extremely high peak of infections in almost all continents at an unprecedented speed and scale. The Omicron variant evades the protection rendered by vaccine-induced antibodies and natural infection, as well as overpowers the antibody-based immunotherapies, raising the concerns of current effectiveness of available vaccines and monoclonal antibody-based therapies. This review outlines the most recent advancements in studying the virology and biology of the Omicron variant, highlighting its increased resistance to current antibody-based therapeutics and its immune escape against vaccines. However, the Omicron variant is highly sensitive to viral fusion inhibitors targeting the HR1 motif in the spike protein, enzyme inhibitors, involving the endosomal fusion pathway, and ACE2-based entry inhibitors. Omicron variant-associated infectivity and entry mechanisms of Omicron variant are essentially distinct from previous characterized variants. Innate sensing and immune evasion of SARS-CoV-2 and T cell immunity to the virus provide new perspectives of vaccine and drug development. These findings are important for understanding SARS-CoV-2 viral biology and advances in developing vaccines, antibody-based therapies, and more effective strategies to mitigate the transmission of the Omicron variant or the next SARS-CoV-2 variant of concern.

An optimized filter design approach for enhancing imaging quality in industrial linear accelerator.

BACKGROUND: The polychromatic X-rays generated by a linear accelerator (Linac) often result in noticeable hardening artifacts in images, posing a significant challenge to accurate defect identification. To address this issue, a simple yet effective approach is to introduce filters at the radiation source outlet. However, current methods are often empirical, lacking scientifically sound metrics. OBJECTIVE: This study introduces an innovative filter design method that optimizes filter performance by balancing the impact of ray intensity and energy on image quality. MATERIALS AND METHODS: Firstly, different spectra under various materials and thicknesses of filters were obtained using GEometry ANd Tracking (Geant4) simulation. Subsequently, these spectra and their corresponding incident photon counts were used as input sources to generate different reconstructed images. By comprehensively comparing the intensity differences and noise in images of defective and non-defective regions, along with considering hardening indicators, the optimal filter was determined. RESULTS: The optimized filter was applied to a Linac-based X-ray computed tomography (CT) detection system designed for identifying defects in graphite materials within high-temperature gas-cooled reactor (HTR), with defect dimensions of 2 mm. After adding the filter, the hardening effect reduced by 22%, and the Defect Contrast Index (DCI) reached 3.226. CONCLUSION: The filter designed based on the parameters of Average Difference (AD) and Defect Contrast Index (DCI) can effectively improve the quality of defect images.

Sertraline Is an Effective SARS-CoV-2 Entry Inhibitor Targeting the Spike Protein.

The global spread of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the continuously emerging new variants underscore an urgent need for effective therapeutics for the treatment of coronavirus disease 2019 (COVID-19). Here, we screened several FDA-approved amphiphilic drugs and determined that sertraline (SRT) exhibits potent antiviral activity against infection of SARS-CoV-2 pseudovirus (PsV) and authentic virus in vitro. It effectively inhibits SARS-CoV-2 spike (S)-mediated cell-cell fusion. SRT targets the early stage of viral entry. It can bind to the S1 subunit of the S protein, especially the receptor binding domain (RBD), thus blocking S-hACE2 interaction and interfering with the proteolysis process of S protein. SRT is also effective against infection with SARS-CoV-2 PsV variants, including the newly emerging Omicron. The combination of SRT and other antivirals exhibits a strong synergistic effect against infection of SARS-CoV-2 PsV. The antiviral activity of SRT is independent of serotonin transporter expression. Moreover, SRT effectively inhibits infection of SARS-CoV-2 PsV and alleviates the inflammation process and lung pathological alterations in transduced mice in vivo. Therefore, SRT shows promise as a treatment option for COVID-19. IMPORTANCE The study shows SRT is an effective entry inhibitor against infection of SARS-CoV-2, which is currently prevalent globally. SRT targets the S protein of SARS-CoV-2 and is effective against a panel of SARS-CoV-2 variants. It also could be used in combination to prevent SARS-CoV-2 infection. More importantly, with long history of clinical use and proven safety, SRT might be particularly suitable to treat infection of SARS-CoV-2 in the central nervous system and optimized for treatment in older people, pregnant women, and COVID-19 patients with heart complications, which are associated with severity and mortality of COVID-19.

Pan-coronavirus fusion inhibitors to combat COVID-19 and other emerging coronavirus infectious diseases.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the currently ongoing coronavirus disease 2019 (COVID-19) pandemic, has posed a serious threat to global public health. Recently, several SARS-CoV-2 variants of concern (VOCs) have emerged and caused numerous cases of reinfection in convalescent COVID-19 patients, as well as breakthrough infections in vaccinated individuals. This calls for the development of broad-spectrum antiviral drugs to combat SARS-CoV-2 and its VOCs. Pan-coronavirus fusion inhibitors, targeting the conserved heptad repeat 1 (HR1) in spike protein S2 subunit, can broadly and potently inhibit infection of SARS-CoV-2 and its variants, as well as other human coronaviruses. In this review, we summarized the most recent development of pan-coronavirus fusion inhibitors, such as EK1, EK1C4, and EKL1C, and highlighted their potential application in combating current COVID-19 infection and reinfection, as well as future emerging coronavirus infectious diseases.

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

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

25-hydroxycholesterol inhibits human papillomavirus infection in cervical epithelial cells by perturbing cytoskeletal remodeling.

Persistent high-risk human papilloma virus (HR-HPV) infection is the main risk factor for cervical cancer, threatening women's health. Despite growing prophylactic vaccination, annual cervical cancer cases are still increasing and show a trend of younger onset age. However, therapeutic approaches towards HPV infection are still limited. 25-hydrocholesterol (25HC) has a wide-spectrum inhibitory effect on a variety of viruses. To explore efficient interventions to restrict HPV infection at an early time, we applied different pseudoviruses (PsV) to evaluate anti-HPV efficacy of 25HC. We tested PsV inhibition by 25HC in cervical epithelial-derived HeLa and C-33A cells, using high-risk (HPV16, HPV18, HPV59), possibly carcinogenic (HPV73), and low-risk (HPV6) HPV PsVs. Then we established murine genital HPV PsV infection models and applied IVIS to evaluate anti-HPV efficacy of 25HC in vivo. Next, with the help of confocal imaging, we targeted 25HC activity at filopodia upon HPV exposure. After that, we used RNA-seq and Western blot analysis to investigate (1) how 25HC disturbs actin cytoskeleton remodeling during HPV infection and (2) how prenylation regulates the cytoskeletal remodeling signaling pathway. Our findings suggest that 25HC perturbs F-actin rearrangement by reducing small GTPase prenylation. In this way, the phenomenon of HPV virion surfing was restricted, leading to failed infection.

SARS-CoV-2 Omicron XBB subvariants exhibit enhanced fusogenicity and substantial immune evasion in elderly population, but high sensitivity to pan-coronavirus fusion inhibitors.

Numerous emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants have shown significant immune evasion capacity and caused a large number of infections, as well as vaccine-breakthrough infections, especially in elderly populations. Recently emerged Omicron XBB was derived from the BA.2 lineage, but bears a distinct mutant profile in its spike (S) protein. In this study, we found that Omicron XBB S protein drove more efficient membrane-fusion kinetics on human lung-derived cells (Calu-3). Considering the high susceptibility of the elderly to the current Omicron pandemic, we performed a comprehensive neutralization assessment of elderly convalescent or vaccine sera against XBB infection. We found that the sera from elderly convalescent patients who experienced with BA.2 infection or breakthrough infection potently inhibited BA.2 infection, but showed significantly reduced efficacy against XBB. Moreover, recently emerged XBB.1.5 subvariant also showed more significant resistance to the convalescent sera of BA.2- or BA.5-infected elderly. On the other hand, we found that the pan-CoV fusion inhibitors EK1 and EK1C4 can potently block either XBB-S- or XBB.1.5-S-mediated fusion process and viral entry. Moreover, EK1 fusion inhibitor showed potent synergism when combined with convalescent sera of BA.2- or BA.5-infected patients against XBB and XBB.1.5 infection, further indicating that EK1-based pan-CoV fusion inhibitors are promising candidates for development as clinical antiviral agents to combat the Omicron XBB subvariants.

Repurposing Navitoclax to block SARS-CoV-2 fusion and entry by targeting heptapeptide repeat sequence 1 in S2 protein.

Along with the long pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has come the dilemma of emerging viral variants of concern (VOC), particularly Omicron and its subvariants, able to deftly escape immune surveillance and the otherwise protective effect of current vaccines and antibody drugs. We previously identified a peptide-based pan-CoV fusion inhibitor, termed as EK1, able to bind the HR1 region in viral spike (S) protein S2 subunit. This effectively blocked formation of the six-helix bundle (6-HB) fusion core and, thus, showed efficacy against all human coronaviruses (HCoVs). EK1 is now in phase 3 clinical trials. However, the peptide drug generally lacks oral availability. Therefore, we herein performed a structure-based virtual screening of the libraries of biologically active molecules and identified nine candidate compounds. One is Navitoclax, an orally active anticancer drug by inhibition of Bcl-2. Like EK1 peptide, it could bind HR1 and block 6-HB formation, efficiently inhibiting fusion and infection of all SARS-CoV-2 variants tested, as well as SARS-CoV and MERS-CoV, with IC50 values ranging from 0.5 to 3.7 µM. These findings suggest that Navitoclax is a promising repurposed drug candidate for development as a safe and orally available broad-spectrum antiviral drug to combat the current SARS-CoV-2 and its variants, as well as other HCoVs.

Characterization of cross-reactive monoclonal antibodies against SARS-CoV-1 and SARS-CoV-2: Implication for rational design and development of pan-sarbecovirus vaccines and neutralizing antibodies.

Emergence of various circulating SARS-CoV-2 variants of concern (VOCs) promotes the identification of pan-sarbecovirus vaccines and broadly neutralizing antibodies (bNAbs). Here, to characterize monoclonal antibodies cross-reactive against both SARS-CoV-1 and SARS-CoV-2 and to search the criterion for bNAbs against all emerging SARS-CoV-2, we isolated several SARS-CoV-1-cross-reactive monoclonal antibodies (mAbs) from a wildtype SARS-CoV-2 convalescent donor. These antibodies showed broad binding capacity and cross-neutralizing potency against various SARS-CoV-2 VOCs, including B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta), but failed to efficiently neutralize Omicron variant and its sublineages. Structural analysis revealed how Omicron sublineages, but not other VOCs, efficiently evade an antibody family cross-reactive against SARS-CoV-1 through their escape mutations. Further evaluation of a series of SARS-CoV-1/2-cross-reactive bNAbs showed a negative correlation between the neutralizing activities against SARS-CoV-1 and SARS-CoV-2 Omicron variant. Together, these results suggest the necessity of using cross-neutralization against SARS-CoV-1 and SARS-CoV-2 Omicron as criteria for rational design and development of potent pan-sarbecovirus vaccines and bNAbs.