Kwok-Hung Chan

In 2024, a bovine H5N1 strain was first isolated from dairy cows in Texas and confirmed to transmit cross-species to humans. Therefore, research on treatments for human infection should be accelerated. In our study, the antiviral effects of baloxavir acid (BXA), oseltamivir carboxylate (OSC), EIDD-1931 (NHC), and ribavirin (RBV) against five H5N1 strains were evaluated in vitro. Cell viability and viral replication were measured to assess the antiviral effects. The results showed that the EC₅₀ of BXA treatment was the lowest. The BXA/NHC and BXA/OSC combination treatments showed more potent inhibitory effects than each monotherapy. The 15 mg/kg baloxavir marboxil (BXM) / 125 mg/kg molnupiravir (MNP) and the 15 mg/kg BXM / 10 mg/kg oseltamivir phosphate (OSP) were tested in BALB/c mice. The mice were inoculated with 10 times the 50% mouse lethal dose (10 MLD₅₀) of bovine H5N1 virus. Treatments began 1-day post-infection (1 dpi) and were administered orally twice daily for 5 or 7 days. Changes in body weight, clinical signs, and survival were monitored; lung and brain tissues were collected for virological, immunological, and histological analyses. Most mice died from severe neurological symptoms. Compared with the 5-day treatment, the 7-day treatment effectively inhibited viral replication and increased survival rates to 50% in BXM, BXM/MNP, and BXM/OSP treatments. Mice treated with BXM/MNP or BXM/OSP combination therapy showed lower viral yields in the lungs than those treated with BXM alone. The results provide a reference for human treatment, and extending the 7-day combination treatment should be considered.

The effectiveness of case isolation and contact quarantine depends critically on the extent of pre-symptomatic transmission. However, the capacity of influenza viruses for transmission before symptom onset remains uncertain. We developed an individual-based household transmission model that explicitly accounts for transmission occurring before symptom onset by marginalizing unobserved infection times. We analyzed influenza household transmission studies of influenza A and B viruses in Hong Kong between 2008 and 2017 (493 and 255 households respectively), estimating pathogen-specific pre-symptomatic transmission proportions and identifying factors affecting individual susceptibility and infectiousness. We estimated that 9.6% (95% credible interval (CrI): 5.9%, 14.7%) of influenza A transmission occurred > 0.5 days earlier than infectors’ symptom onset, while influenza B showed no evidence of pre-symptomatic transmission. Younger age was associated with an increased susceptibility and infectiousness for both influenza A and B viruses. Influenza A infectiousness was positively associated with viral load and fever. Using an additional SARS-CoV-2 Omicron dataset in Hong Kong, our analysis confirmed substantial pre-symptomatic transmission (65.5%, 95% CrI: 52.7%, 78.9%), validating our methodology. Our modeling framework provides a robust approach for quantifying pre-symptomatic transmission across respiratory pathogens in households, offering a methodological template for resolving similar empirical uncertainties in infectious disease transmission. Our findings suggest symptom-based control measures may be feasible for influenza, though quarantine of asymptomatic contacts would provide limited additional benefit.

H9N2 is currently the second most common avian influenza A virus subtype infecting humans. Monitoring viral phenotypic and genotypic adaptation to humans is crucial for risk assessment. Here, we compared the replication of an H9N2 human isolate collected in 2024 (A/HK/2346/2024) to a historical human isolate collected in 1999 (A/HK/1073/1999). In Madin Darby canine kidney (MDCK) cells, A/HK/2346/2024 and A/HK/1073/1999 replicated to 8 log10 and 5 log10 plaque-forming units (PFU) per ml, respectively. In both human nasal and lung organoids, A/HK/2346/2024 replicated to 6 log10 PFU/ml, but A/HK/1073/1999 failed to replicate in either organoid. The infection rates of both ciliated and non-ciliated cells were higher for A/HK/2346/2024 than A/HK/1073/1999, and the ratios of infected 2,6/2,3 cells were also higher for A/HK/2346/2024. Apart from the mammalian adaptive substitutions that were present in the nasopharyngeal specimen collected on day 1 post-symptom onset (pso) (HA-D183N/D190 T/Q192R/Q226L; NA-del62-64; PB2-A588 V/K702R; PB1-I368 V; PA-K356R/S409N; M1-R95 K), the mammalian adaptive substitution PB2-D253N emerged de novo on day 7 pso. Analysis of all human (n = 96) and avian influenza (n = 14762) H9N2 deposited at GISAID showed the dominance of several human-adaptation substitutions in H9N2 strains collected from humans in recent years. In summary, we demonstrated that a recent H9N2 virus is more adapted to humans, and is able to replicate to high titers in both upper and lower human respiratory tract which may confer higher person-to-person transmissibility and virulence. Our study underscores the importance of human organoid-based phenotypic monitoring and inter/intrahost genotypic monitoring for assessing the zoonotic risk of avian influenza viruses.

Gold standard detection of SARS-CoV-2 by reverse transcription quantitative PCR (RT-qPCR) can achieve ultrasensitive viral detection down to a few RNA copies per sample. Yet, the lengthy detection and labor-intensive protocol limit its effectiveness in community screening. In view of this, a structural switching electrochemical aptamer-based biosensor (E-AB) targeting the SARS-CoV-2 nucleocapsid (N) protein was developed. Four N protein-targeting aptamers were characterized on an electrochemical cell configuration using square wave voltammetry (SWV). The sensor was investigated in an artificial saliva matrix optimizing the aptamer anchoring orientation, SWV interrogation frequency, and target incubation time. Rapid detection of the N protein was achieved within 5 min at a low nanomolar limit of detection (LOD) with high specificity. Specific N protein detection was also achieved in simulated positive saliva samples, demonstrating its feasibility for saliva-based rapid diagnosis. Further research will incorporate novel signal amplification strategies to improve sensitivity for early diagnosis.

Background: This study explored neutralizing IgG antibody levels against COVID-19 decline over time post-vaccination. We conducted this prospective cohort study to investigate the function of gut microbiota in the host immune response following three doses of BNT162b2. Methods: Subjects who received three doses of BNT162b2 were recruited from three centers in Hong Kong. Blood samples were obtained before the first dose and at the one-year timepoint for IgG ELISA to determine the level of neutralizing antibody (NAb). The primary outcome was a high immune response (NAb > 600 AU/mL). We performed shotgun DNA metagenomic sequencing on baseline fecal samples to identify bacterial species and metabolic pathways associated with high immune response using linear discriminant analysis effect size analysis. Results: A total of 125 subjects were recruited (median age: 52 years [IQR: 46.2–59.0]; male: 43 [34.4%]), and 20 were regarded as low responders at the one-year timepoint. Streptococcus parasanguinis (log₁₀LDA score = 2.38, p = 0.003; relative abundance of 2.97 × 10⁻⁵ vs. 0.03%, p = 0.001), Bacteroides stercoris (log₁₀LDA score = 4.29, p = 0.024; relative abundance of 0.14% vs. 2.40%, p = 0.014) and Haemophilus parainfluenzae (log₁₀LDA score = 2.15, p = 0.022; relative abundance of 0.01% vs. 0, p = 0.010) were enriched in low responders. Bifidobacterium pseudocatenulatum (log₁₀LDA score = 2.99, p = 0.048; relative abundance of 0.09% vs. 0.36%, p = 0.049) and Clostridium leptum (log₁₀LDA score = 2.38, p = 0.014; relative abundance of 1.2 × 10⁻⁵% vs. 0, p = 0.044) were enriched in high responders. S. parasanguinis was negatively correlated with the superpathway of pyrimidine ribonucleotides de novo biosynthesis (log₁₀LDA score = 2.63), which contributes to inflammation and antibody production. H. parainfluenzae was positively correlated with pathways related to anti-inflammatory processes, including the superpathway of histidine, purine, and pyrimidine biosynthesis (log₁₀LDA score = 2.14). Conclusion: Among three-dose BNT162b2 recipients, S. parasanguinis, B. stercoris and H. parainfluenzae were associated with poorer immunogenicity at one year, while B. pseudocatenulatum and C. leptum was associated with a better response.

Background: Neutralizing antibody level wanes with time after COVID-19 vaccination. We aimed to study the relationship between baseline gut microbiota and immunogenicity after three doses of CoronaVac. Methods: This was a prospective cohort study recruiting three-dose CoronaVac recipients from two centers in Hong Kong. Blood samples were collected at baseline and one year post-first dose for virus microneutralization (vMN) assays to determine neutralization titers. The primary outcome was high immune response (defined as with vMN titer ≥ 40). Shotgun DNA metagenomic sequencing of baseline fecal samples identified potential bacterial species and metabolic pathways using Linear Discriminant Analysis Effect Size (LEfSe) analysis. Univariate and multivariable logistic regression models were used to identify high response predictors. Results: In total, 36 subjects were recruited (median age: 52.7 years [IQR: 47.9–56.4]; male: 14 [38.9%]), and 18 had low immune response at one year post-first dose vaccination. Eubacterium rectale (log₁₀LDA score = 4.15, p = 0.001; relative abundance of 1.4% vs. 0, p = 0.002), Collinsella aerofaciens (log₁₀LDA score = 3.31, p = 0.037; 0.39% vs. 0.18%, p = 0.038), and Streptococcus salivarius (log₁₀LDA score = 2.79, p = 0.021; 0.05% vs. 0.02%, p = 0.022) were enriched in low responders. The aOR of high immune response with E. rectale, C. aerofaciens, and S. salivarius was 0.03 (95% CI: 9.56 × 10⁻⁴–0.32), 0.03 (95% CI: 4.47 × 10⁻⁴–0.59), and 10.19 (95% CI: 0.81–323.88), respectively. S. salivarius had a positive correlation with pathways enriched in high responders like incomplete reductive TCA cycle (log₁₀LDA score = 2.23). C. aerofaciens similarly correlated with amino acid biosynthesis-related pathways. These pathways all showed anti-inflammation functions. Conclusion: E. rectale,C. aerofaciens, and S. salivarius correlated with poorer long-term immunogenicity following three doses of CoronaVac.

CoronaVac immunogenicity decreases with time, and we aimed to investigate whether gut microbiota associate with longer-term immunogenicity of CoronaVac. This was a prospective cohort study recruiting two-dose CoronaVac recipients from three centres in Hong Kong. We collected blood samples at baseline and day 180 after the first dose and used chemiluminescence immunoassay to test for neutralizing antibodies (NAbs) against the receptor-binding domain (RBD) of wild-type SARS-CoV-2 virus. We performed shotgun metagenomic sequencing performed on baseline stool samples. The primary outcome was the NAb seroconversion rate (seropositivity defined as NAb ≥ 15AU/mL) at day 180. Linear discriminant analysis [LDA] effect size analysis was used to identify putative bacterial species and metabolic pathways. A univariate logistic regression model was used to derive the odds ratio (OR) of seropositivity with bacterial species. Of 119 CoronaVac recipients (median age: 53.4 years [IQR: 47.8–61.3]; male: 39 [32.8%]), only 8 (6.7%) remained seropositive at 6 months after vaccination. Bacteroides uniformis (log₁₀LDA score = 4.39) and Bacteroides eggerthii (log₁₀LDA score = 3.89) were significantly enriched in seropositive than seronegative participants. Seropositivity was associated with B. eggerthii (OR: 5.73; 95% CI: 1.32–29.55; p = 0.022) and B. uniformis with borderline significance (OR: 3.27; 95% CI: 0.73–14.72; p = 0.110). Additionally, B. uniformis was positively correlated with most enriched metabolic pathways in seropositive vaccinees, including the superpathway of adenosine nucleotide de novo biosynthesis I (log₁₀LDA score = 2.88) and II (log₁₀LDA score = 2.91), as well as pathways related to vitamin B biosynthesis, all of which are known to promote immune functions. In conclusion, certain gut bacterial species (B. eggerthii and B. uniformis) and metabolic pathways were associated with longer-term CoronaVac immunogenicity.

Background: Mpox virus (MPXV), previously known as monkeypox virus, has spread globally in 2022. An accurate and convenient antibody test is essential for the determination of seroprevalence and for studying immune response after natural infection or vaccination. Most seroprevalence or vaccine studies used either live MPXV (or vaccinia virus [VACV]) or inactivated MPXV (or VACV) culture lysate for serological assays, but MPXV culture can only be performed in biosafety level 3 (BSL-3) facilities. Here, we developed and evaluated an enzyme immunoassay (EIA) based on the MPXV A29 surface envelope protein. Methods: We compared the specificity of the MPXV A29, VACV A27, and VACV lysate EIA using serum specimens collected prior to the global spread of MPXV. Next, we performed these EIAs for serum specimens collected from two mpox patients and an MVA-BN vaccine recipient. We also assessed the kinetics of plasmblast and MPXV A29-specific B-cell response. Results: Using sera collected from different age groups in Hong Kong, we found that most individuals, including those born before 1981 who have received the smallpox vaccine, tested negative using the MPXV A29 protein. MPXV A29-specific antibody could be detected in the serum of mpox patients and an MVA-BN recipient. In a mpox patient, the frequency of plasmablast and MPXV A29-specific B cell peaked on day 8 post-symptom onset and gradually decreased. Finally, we demonstrated that antibodies against the A29 protein can be used for immunofluorescence staining of MPXV-infected cells. Conclusions: MPXV A29 protein is suitable for studying the immune response against MPXV infection.