Kwok-Yung Yuen’s research while affiliated with The University of Hong Kong and other places

The highly pathogenic avian influenza (HPAI) H5N1 virus has been endemic in aquatic birds since 1997, causing outbreaks in domestic poultry and occasional human infections worldwide. Recently, the cross-species transmission of a new reassortant variant from clade 2.3.4.4b of H5N1 to cattle in the US has heightened concerns regarding the expansion of host range and potential human infection. As eradicating the H5N1 virus from its reservoir is impossible, it is essential to prepare for a potential pandemic caused by an H5N1 derivative. Utilizing a deleted-NS1 live attenuated influenza viral vector vaccine system (DelNS1 LAIV), a system we have previously used in the development of a COVID-19 vaccine, we have rapidly developed an intranasal vaccine for cattle H5N1 and related clade 2.3.4.4b strains, based on publicly available sequences. Our research demonstrates that a single intranasal immunization can provide effective protection against lethal challenges from HPAI cattle or mink H5N1 variants, offering strong, sustained immunity after two months in female mouse and male hamster models. Immunogenicity analysis reveals that intranasal vaccination with DelNS1 LAIV induces robust neutralizing antibody, mucosal IgA and T cell responses in mice. It is crucial to further evaluate the DelNS1-H5N1 LAIV system to prepare for potential future H5N1 outbreaks in humans.

SARS-CoV-2 JN.1 with an additional L455S mutation on spike when compared with its parental variant BA.2.86 has outcompeted all earlier variants to become the dominant circulating variant. Recent studies investigated the immune resistance of SARS-CoV-2 JN.1 but additional factors are speculated to contribute to its global dominance, which remain elusive until today. Here, we find that SARS-CoV-2 JN.1 has a higher infectivity than BA.2.86 in differentiated primary human nasal epithelial cells (hNECs). Mechanistically, we demonstrate that the gained infectivity of SARS-CoV-2 JN.1 over BA.2.86 associates with increased entry efficiency conferred by L455S and better spike cleavage in hNECs. Structurally, S455 altered the mode of binding of JN.1 spike protein to ACE2 when compared to BA.2.86 spike at ACE2H34, and modified the internal structure of JN.1 spike protein by increasing the number of hydrogen bonds with neighboring residues. These findings indicate that a single mutation (L455S) enhances virus entry in hNECs and increases immune evasiveness, which contribute to the robust transmissibility of SARS-CoV-2 JN.1. We further evaluate the in vitro and in vivo virological characteristics between SARS-CoV-2 BA.2.86/JN.1 and EG.5.1/HK.3, and identify key lineage-specific features of the two Omicron sublineages that contribute to our understanding on Omicron antigenicity, transmissibility, and pathogenicity.

Background The spread of emerging SARS-CoV-2 immune escape sublineages, especially JN.1 and KP.2, has resulted in new waves of COVID-19 globally. The evolving memory B cell responses elicited by the parental Omicron variants to subvariants with substantial antigenic drift remain incompletely investigated. Methods Using the single B cell antibody cloning technology, we isolated single memory B cells, delineated the B cell receptor repertoire and conducted the pseudovirus-based assay for recovered neutralizing antibodies (NAb) screening. We analyzed the cryo-EM structures of top broadly NAbs (bnAbs) and evaluated their in vivo efficacy (golden Syrian hamster model). Findings By investigating the evolution of human B cell immunity, we discovered a new panel of bnAbs arising from vaccinees after Omicron BA.2/BA.5 breakthrough infections. Two lead bnAbs neutralized major Omicron subvariants including JN.1 and KP.2 with IC50 values less than 10 ng/mL, representing ultrapotent receptor binding domain (RBD)-specific class I bnAbs. They belonged to the IGHV3-53/3-66 clonotypes instead of evolving from the pre-existing vaccine-induced IGHV1-58/IGKV3-20 bnAb ZCB11. Despite sequence diversity, they targeted previously unrecognized, highly conserved conformational epitopes in the receptor binding motif (RBM) for ultrapotent ACE2 blockade. The lead bnAb ZCP3B4 not only protected the lungs of hamsters intranasally challenged with BA.5.2, BQ.1.1 and XBB.1.5 but also prevented their contact transmission. Interpretation Our findings demonstrated that class I bnAbs have evolved an ultrapotent mode of action protecting against highly transmissible and broad Omicron escape variants, and their epitopes are potential targets for novel bnAbs and vaccine development. Funding A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Background Post-acute sequalae of COVID-19 defines a wide range of ongoing symptoms and conditions long after SARS-CoV-2 infection including respiratory diseases. The histopathological changes in the lung and underlying mechanism remain elusive. Methods We investigated lung histopathological and transcriptional changes in SARS-CoV-2-infected male hamsters at 7, 14, 42, 84 and 120dpi, and compared with A (H1N1)pdm09 infection. Findings We demonstrated viral residue, inflammatory and fibrotic changes in lung after SARS-CoV-2 but not H1N1 infection. The most prominent histopathological lesion was multifocal alveolar-bronchiolization observed in every SARS-CoV-2 infected hamster (31/31), from 42dpi to 120dpi. Proliferating (Ki67+) CK14+ basal cells accumulated in alveoli adjacent to bronchioles at 7dpi, where they proliferated and differentiated into SCGB1A+ club cell or Tubulin+ ciliated cells forming alveolar-bronchiolization foci. Molecularly, Notch pathway significantly upregulated with intensive Notch3 and Hes1 protein expression in alveolar-bronchiolization foci at 42 and 120dpi, suggesting Notch signaling involving the persistence of alveolar-bronchiolization. This is further demonstrated by spatial transcriptomic analysis. Intriguingly, significant upregulation of some cell-growth promoting pathways and genes such as Tubb4b, Stxbp4, Grb14 and Mlf1 were spatially overlapping with bronchiolization lesion. Interpretation Incomplete resolution of SARS-CoV-2 infection in lung with viral residue, chronic inflammatory and fibrotic damage and alveolar-bronchiolization impaired respiratory function. Aberrant activation of CK14+ basal cells during tissue regeneration led to persistent alveolar-bronchiolization due to sustained Notch signaling. This study advances our understanding of respiratory PASC, sheds light on disease management and highlights the necessity for monitoring disease progression in people with respiratory PASC. Funding Funding is listed in the Acknowledgements section.

Hepatitis E virus (HEV) variants infecting humans belong to two species: Paslahepevirus balayani (bHEV) and Rocahepevirus ratti (rat hepatitis E virus; rHEV). R. ratti is a ubiquitous rodent pathogen that has recently been recognized to cause hepatitis in humans. Transmission routes of rHEV from rats to humans are currently unknown. In this study, we examined rHEV exposure in cats and dogs to determine if they are potential reservoirs of this emerging human pathogen. Virus-like particle-based IgG enzymatic immunoassays (EIAs) capable of differentiating rHEV & bHEV antibody profiles and rHEV-specific real-time RT-PCR assays were used for this purpose. The EIAs could detect bHEV and rHEV patient-derived IgG spiked in dog and cat sera. Sera from 751 companion dogs and 130 companion cats in Hong Kong were tested with these IgG enzymatic immunoassays (EIAs). Overall, 13/751 (1.7%) dogs and 5/130 (3.8%) cats were sero-reactive to HEV. 9/751 (1.2%) dogs and 2/130 (1.5%) cats tested positive for rHEV IgG, which was further confirmed by rHEV immunoblots. Most rHEV-seropositive animals were from areas in or adjacent to districts reporting human rHEV infection. Neither 881 companion animals nor 652 stray animals carried rHEV RNA in serum or rectal swabs. Therefore, we could not confirm a role for cats and dogs in transmitting rHEV to humans. Further work is required to understand the reasons for low-level seropositivity in these animals.

Host survival depends on the elimination of virus and mitigation of tissue damage. Herein, we report the modulation of D-mannose flux rewires the virus-triggered immunometabolic response cascade and reduces tissue damage. Safe and inexpensive D-mannose can compete with glucose for the same transporter and hexokinase. Such competitions suppress glycolysis, reduce mitochondrial reactive-oxygen-species and succinate-mediated hypoxia-inducible factor-1α, and thus reduce virus-induced proinflammatory cytokine production. The combinatorial treatment by D-mannose and antiviral monotherapy exhibits in vivo synergy despite delayed antiviral treatment in mouse model of virus infections. Phosphomannose isomerase (PMI) knockout cells are viable, whereas addition of D-mannose to the PMI knockout cells blocks cell proliferation, indicating that PMI activity determines the beneficial effect of D-mannose. PMI inhibition suppress a panel of virus replication via affecting host and viral surface protein glycosylation. However, D-mannose does not suppress PMI activity or virus fitness. Taken together, PMI-centered therapeutic strategy clears virus infection while D-mannose treatment reprograms glycolysis for control of collateral damage.

Developing recombinant proteins as nasal vaccines for inducing systemic and mucosal immunity against respiratory viruses is promising. However, additional adjuvants are required to overcome the low immunogenicity of protein antigens. Here, a self-adjuvanted protein-RNA ribonucleoprotein vaccine was developed and found to be an effective nasal vaccine in mice and the SARS-CoV-2 infection model. The vaccine consisted of spike RBD (as an antigen), nucleoprotein (as an adaptor), and ssRNA (as an adjuvant and RNA scaffold). This combination robustly induced mucosal IgA, neutralizing antibodies and activated multifunctional T-cells, while also providing sterilizing immunity against live virus challenge. In addition, high-resolution scRNA-seq analysis highlighted airway-resident immune cells profile during prime-boost immunization. The vaccine also possesses modularity (antigen/adaptor/RNA scaffold) and can be made to target other viruses. This protein-RNA ribonucleoprotein vaccine is a novel and promising approach for developing safe and potent nasal vaccines to combat respiratory virus infections.

Staphylococcus argenteus is a novel Staphylococcus species derived from Staphylococcus aureus. Information on the prevalence and genetic characteristics of invasive S. argenteus in Asia is limited. In this study, 275 invasive S. aureus complex strains were retrieved from blood culture specimens in Hong Kong and re-analyzed using MALDI-TOF mass spectrometry and an in-house multiplex real-time PCR for S. argenteus. The prevalence of invasive S. argenteus in Hong Kong was found to be 4.0% (11/275). These strains were primarily susceptible to commonly used antibiotics, except penicillin. Whole-genome sequencing revealed the circulation of three S. argenteus genotypes (ST-2250, ST-1223, and ST-2854) in Hong Kong, with ST-2250 and ST-1223 being the predominant genotypes. The local ST-2250 and ST-1223 strains showed close phylogenetic relationships with isolates from mainland China. Antimicrobial-resistant genes (fosB, tet-38, mepA, blaI, blaZ) could be found in nearly all local S. argenteus strains. The ST-1223 and ST-2250 genotypes carried multiple staphylococcal enterotoxin genes that could cause food poisoning and toxic shock syndrome. The CRISPR/Cas locus was observed only in the ST-2250 strains. This study provides the first report on the molecular epidemiology of invasive S. argenteus in Hong Kong, and further analysis is needed to understand its transmission reservoir.

Introduction Therapeutic monoclonal antibodies (mAbs) against the SARS-CoV-2 spike protein have been shown to improve the outcome of severe COVID-19 patients in clinical trials. However, novel variants with spike protein mutations can render many currently available mAbs ineffective. Methods We produced mAbs by using hybridoma cells that generated from mice immunized with spike protein trimer and receptor binding domain (RBD). The panel of mAbs were screened for binding and neutralizing activity against different SARS-CoV-2 variants. The in vivo effectiveness of WKS13 was evaluated in a hamster model. Results Out of 960 clones, we identified 18 mAbs that could bind spike protein. Ten of the mAbs could attach to RBD, among which five had neutralizing activity against the ancestral strain and could block the binding between the spike protein and human ACE2. One of these mAbs, WKS13, had broad neutralizing activity against all Variants of Concern (VOCs), including the Omicron variant. Both murine or humanized versions of WKS13 could reduce the lung viral load in hamsters infected with the Delta variant. Conclusions Our data showed that broad-spectrum high potency mAbs can be produced from immunized mice, which can be used in humans after humanization of the Fc region. Our method represents a versatile and rapid strategy for generating therapeutic mAbs for upcoming novel variants.

Background: Among the Omicron sublineages that have emerged, BA.1, BA.2, BA.5, and their related sublineages have resulted in the largest number of infections. While recent studies demonstrated that all Omicron sublineages robustly escape neutralizing antibody response, it remains unclear on whether these Omicron sublineages share any pattern of evolutionary trajectory on their replication efficiency and intrinsic pathogenicity along the respiratory tract. Methods: We compared the virological features, replication capacity of dominant Omicron sublineages BA.1, BA.2 and BA.5 in the human nasal epithelium, and characterized their pathogenicity in K18-hACE2, A129, young C57BL/6, and aged C57BL/6 mice. Findings: We found that BA.5 replicated most robustly, followed by BA.2 and BA.1, in the differentiated human nasal epithelium. Consistently, BA.5 infection resulted in higher viral gene copies, infectious viral titres and more abundant viral antigen expression in the nasal turbinates of the infected K18-hACE2 transgenic mice. In contrast, the Omicron sublineages are continuously attenuated in lungs of infected K18-hACE2 and C57BL/6 mice, leading to decreased pathogenicity. Nevertheless, lung manifestations remain severe in Omicron sublineages-infected A129 and aged C57BL/6 mice. Interpretation: Our results suggested that the Omicron sublineages might be gaining intrinsic replication fitness in the upper respiratory tract, therefore highlighting the importance of global surveillance of the emergence of hyper-transmissive Omicron sublineages. On the contrary, replication and intrinsic pathogenicity of Omicron is suggested to be further attenuated in the lower respiratory tract. Effective vaccination and other precautions should be in place to prevent severe infections in the immunocompromised populations at risk. Funding: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.