Publications by Year: 2022

DC-SIGN and Galectin-3 are two different lectins and have been reported to participate in regulation of several virus infections. WHO has pointed that H5N1 and H7N9 avian influenza viruses (AIVs) play continuous threats to global health. AIV hemagglutinin (HA) protein, a highly glycosylated protein, mediated influenza infection and was proposed to have DC-SIGN and Gal3 interactive domains. This study aims to address the individual and collaborative roles of DC-SIGN and Gal3 toward AIVs infection. Firstly, A549 cells with DC-SIGN expression or Gal3-knockdown, via lentiviral vector-mediated CD209 gene expression or LGALS-3 gene knockdown, respectively were generated. Quantitative Reverse Transcription PCR (qRT-PCR) results indicated that DC-SIGN expression and Gal3 knockdown in A549 cells were significantly promoted and ameliorated HA or NP gene expression, respectively after H5N1 and H7N9-reverse genetics (RG) virus post-infections (P < 0.05). Similar results observed in immunoblotting, indicating that DC-SIGN expression significantly facilitated H5N1-RG and H7N9-RG infections (P < 0.05) whereas Gal3 knockdown significantly reduced both viral infections (P < 0.05). Furthermore, we found that DC-SIGN and Gal3 co-expression significantly enhanced infectivity of both H5N1-RG and H7N9-RG viruses (P < 0.01) and higher regulatory capabilities by DC-SIGN and Gal3 in H5N1-RG than H7N9-RG was noted. The promoting effect mainly relied on exogenous Gal3 and DC-SIGN directly interacting with the HA protein of H5N1 or H7N9 AIVs, subsequently enhancing virus infection. This study sheds light on two different lectins individually and collaboratively regulating H5N1 and H7N9 AIVs infection and the inhibitors against DC-SIGN and Gal3 interacting with HA could be utilized as alternative antiviral strategies.

The interaction of SARS-CoV-2 infection with extracellular vesicles (EVs) is of particular interest at the moment. Studying SARS-CoV-2 contaminated-EV isolates in instruments located outside of the biosafety level-3 (BSL-3) environment requires knowing how viral inactivation methods affect the structure and function of extracellular vesicles (EVs). Therefore, three common viral inactivation methods, ultraviolet-C (UVC; 1350 mJ/cm²), β-propiolactone (BPL; 0.005%), heat (56°C, 45 min) were performed on defined EV particles and their proteins, RNAs, and function. Small EVs were isolated from the supernatant of SARS-CoV-2-infected human lung epithelial Calu-3 cells by stepwise centrifugation, ultrafiltration and qEV size-exclusion chromatography. The EV isolates contained SARS-CoV-2. UVC, BPL and heat completely abolished SARS-CoV-2 infectivity of the contaminated EVs. Particle detection by electron microscopy and nanoparticle tracking was less affected by UVC and BPL than heat treatment. Western blot analysis of EV markers was not affected by any of these three methods. UVC reduced SARS-CoV-2 spike detectability by quantitative RT-PCR and slightly altered EV-derived β-actin detection. Fibroblast migration-wound healing activity of the SARS-CoV-2 contaminated-EV isolate was only retained after UVC treatment. In conclusion, specific viral inactivation methods are compatible with specific measures in SARS-CoV-2 contaminated-EV isolates. UVC treatment seems preferable for studying functions of EVs released from SARS-CoV-2 infected cells.

Coronavirus disease 2019 (COVID-19) is an acute respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The prevention of SARS-CoV-2 transmission has become a global priority. Previously, we showed that a protein subunit vaccine that was developed based on the fusion of the SARS-CoV-2 receptor-binding domain (RBD) to the Fc portion of human IgG1 (RBD-Fc), produced in Nicotiana benthamiana, and adjuvanted with alum, namely, Baiya SARS-CoV-2 Vax 1, induced potent immunological responses in both mice and cynomolgus monkeys. Hence, this study evaluated the protective efficacy, safety, and toxicity of Baiya SARS-CoV-2 Vax 1 in K18-hACE2 mice, monkeys and Wistar rats. Two doses of vaccine were administered three weeks apart on Days 0 and 21. The administration of the vaccine to K18-hACE2 mice reduced viral loads in the lungs and brains of the vaccinated animals and protected the mice against challenge with SARS-CoV-2. In monkeys, the results of safety pharmacology tests, general clinical observations, and a core battery of studies of three vital systems, namely, the central nervous, cardiovascular, and respiratory systems, did not reveal any safety concerns. The toxicology study of the vaccine in rats showed no vaccine-related pathological changes, and all the animals remained healthy under the conditions of this study. Furthermore, the vaccine did not cause any abnormal toxicity in rats and was clinically tolerated even at the highest tested concentration. In addition, general health status, body temperature, local toxicity at the administration site, hematology, and blood chemistry parameters were also monitored. Overall, this work presents the results of the first systematic study of the safety profile of a plant-derived vaccine, Baiya SARS-CoV-2 Vax 1; this approach can be considered a viable strategy for the development of vaccines against COVID-19.

Effective mRNA SARS-CoV-2 vaccines are available but need to be stored in freezers, limiting their use to countries that have appropriate storage capacity. ChulaCov19 is a prefusion non-stabilized SARS-CoV-2 spike-protein-encoding, nucleoside-modified mRNA, lipid nanoparticle encapsulated vaccine that we report to be stable when stored at 2–8 °C for up to 3 months. Here we report safety and immunogenicity data from a phase I open-label, dose escalation, first-in-human trial of the ChulaCov19 vaccine (NCT04566276). Seventy-two eligible volunteers, 36 of whom were aged 18–55 (adults) and 36 aged 56–75 (elderly), were enroled. Two doses of vaccine were administered 21 d apart at 10, 25 or 50 μg per dose (12 per group). The primary outcome was safety and the secondary outcome was immunogenicity. All three dosages of ChulaCov19 were well tolerated and elicited robust dose-dependent and age-dependent B- and T-cell responses. Transient mild/moderate injection site pain, fever, chills, fatigue and headache were more common after the second dose. Four weeks after the second dose, in the adult cohort, MicroVNT-50 geometric mean titre against wild-type SARS-CoV-2 was 848 (95% CI, 483–1,489), 736 (459–1,183) and 1,140 (854–1,522) IU ml⁻¹ at 10, 25 and 50 μg doses, respectively, versus 285 (196–413) IU ml⁻¹ for human convalescent sera. All dose levels elicited 100% seroconversion, with geometric mean titre ratios 4–8-fold higher than for human convalescent sera (P < 0.01), and high IFNγ spot-forming cells per million peripheral blood mononuclear cells. The 50 μg dose induced better cross-neutralization against Alpha, Beta, Gamma and Delta variants than lower doses. ChulaCov19 at 50 μg is well tolerated and elicited higher neutralizing antibodies than human convalescent sera, with strong T-cell responses. These antibodies cross-neutralized four variants of concern. ChulaCov19 has proceeded to phase 2 clinical trials. We conclude that the mRNA vaccine expressing a prefusion non-stabilized spike protein is safe and highly immunogenic.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus emerged in late 2019 leading to the COVID-19 disease pandemic that triggered socioeconomic turmoil worldwide. A precise, prompt, and affordable diagnostic assay is essential for the detection of SARS-CoV-2 as well as its variants. Antibody against SARS-CoV-2 spike (S) protein was reported as a suitable strategy for therapy and diagnosis of COVID-19. We, therefore, developed a quick and precise phase-sensitive surface plasmon resonance (PS-SPR) biosensor integrated with a novel generated anti-S monoclonal antibody (S-mAb). Our results indicated that the newly generated S-mAb could detect the original SARS-CoV-2 strain along with its variants. In addition, a SARS-CoV-2 pseudovirus, which could be processed in BSL-2 facility was generated for evaluation of sensitivity and specificity of the assays including PS-SPR, homemade target-captured ELISA, spike rapid antigen test (SRAT), and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Experimentally, PS-SPR exerted high sensitivity to detect SARS-CoV-2 pseudovirus at 589 copies/ml, with 7-fold and 70-fold increase in sensitivity when compared with the two conventional immunoassays, including homemade target-captured ELISA (4 × 10³ copies/ml) and SRAT (4 × 10⁴ copies/ml), using the identical antibody. Moreover, the PS-SPR was applied in the measurement of mimic clinical samples containing the SARS-CoV-2 pseudovirus mixed with nasal mucosa. The detection limit of PS-SPR is calculated to be 1725 copies/ml, which has higher accuracy than homemade target-captured ELISA (4 × 10⁴ copies/ml) and SRAT (4 × 10⁵ copies/ml) and is comparable with qRT-PCR (1250 copies/ml). Finally, the ability of PS-SPR to detect SARS-CoV-2 in real clinical specimens was further demonstrated, and the assay time was less than 10 min. Taken together, our results indicate that this novel S-mAb integrated into PS-SPR biosensor demonstrates high sensitivity and is time-saving in SARS-CoV-2 virus detection. This study suggests that incorporation of a high specific recognizer in SPR biosensor is an alternative strategy that could be applied in developing other emerging or re-emerging pathogenic detection platforms.

Establishment of an mRNA vaccine platform in low- and middle-income countries (LMICs) is important to enhance vaccine accessibility and ensure future pandemic preparedness. Here, we describe the preclinical studies of a SARS-CoV-2 mRNA encoding prefusion-unstabilized ectodomain spike protein encapsulated in lipid nanoparticles (LNP) “ChulaCov19”. In BALB/c mice, ChulaCov19 at 0.2, 1, 10, and 30 μg given 2 doses, 21 days apart, elicited robust neutralizing antibody (NAb) and T cells responses in a dose-dependent relationship. The geometric mean titer (GMT) of micro-virus neutralizing (micro-VNT) antibody against wild-type virus was 1,280, 11,762, 54,047, and 62,084, respectively. Higher doses induced better cross-neutralizing antibody against Delta and Omicron variants. This elicited specific immunogenicity was significantly higher than those induced by homologous prime-boost with inactivated (CoronaVac) or viral vector (AZD1222) vaccine. In heterologous prime-boost study, mice primed with either CoronaVac or AZD1222 vaccine and boosted with 5 μg ChulaCov19 generated NAb 7-fold higher against wild-type virus (WT) and was also significantly higher against Omicron (BA.1 and BA.4/5) than homologous CoronaVac or AZD1222 vaccination. AZD1222-prime/mRNA-boost had mean spike-specific IFNγ positive T cells of 3,725 SFC/10⁶ splenocytes, which was significantly higher than all groups except homologous ChulaCov19. Challenge study in human-ACE-2-expressing transgenic mice showed that ChulaCov19 at 1 μg or 10 μg protected mice from COVID-19 symptoms, prevented SARS-CoV-2 viremia, significantly reduced tissue viral load in nasal turbinate, brain, and lung tissues 99.9-100%, and without anamnestic of Ab response which indicated its protective efficacy. ChulaCov19 is therefore a promising mRNA vaccine candidate either as a primary or a boost vaccination and has entered clinical development.

We studied the virucidal efficacy of 0.4% povidone-iodine (PVP-I) nasal spray against SARS-CoV-2 in the patients’ nasopharynx at 3 minutes and 4 hours after PVP-I exposure. We used an open-label, before and after design, single arm pilot study of adult patients with RT-PCR-confirmed COVID-19 within 24 hours. All patients received three puffs of 0.4% PVP-I nasal spray in each nostril. Nasopharyngeal (NP) swabs were collected before the PVP-I spray (baseline, left NP samples), and at 3 minutes (left and right NP samples) and 4 hours post-PVP-I spray (right NP samples). All swabs were coded to blind assessors and transported to diagnostic laboratory and tested by RT-PCR and cultured to measure the viable SARS-CoV-2 within 24 hours after collection. Fourteen patients were enrolled but viable SARS-CoV-2 was cultured from 12 patients (85.7%). The median viral titer at baseline was 3.5 log TCID₅₀/mL (IQR 2.8-4.0 log TCID₅₀/mL). At 3 minutes post-PVP-I spray via the left nostril, viral titers were reduced in 8 patients (66.7%). At 3 minutes post-PVP-I, the median viral titer was 3.4 log TCID₅₀/mL (IQR 1.8-4.4 log TCID₅₀/mL) (P=0.162). At 4 hours post-PVP-I spray via the right nostril, 6 of 11 patients (54.5%) had either the same or minimal change in viral titers. The median viral titer 3 minutes post-PVP-I spray was 2.7 log TCID₅₀/mL (IQR 2.0-3.9 log TCID₅₀/mL). Four hours post-PVP-I spray the median titer was 2.8 log TCID₅₀/mL (IQR 2.2-3.9 log TCID₅₀/mL) (P=0.704). No adverse effects of 0.4% PVP-I nasal spray were detected. We concluded that 0.4% PVP-I nasal spray demonstrated minimal virucidal efficacy at 3 minutes post-exposure. At 4 hours post-exposure, the viral titer was considerably unchanged from baseline in 10 cases. The 0.4% PVP-I nasal spray showed poor virucidal activity and is unlikely to reduce transmission of SARS-CoV-2 in prophylaxis use.

Viral assembly and budding are the final steps and key determinants of the virus life cycle and are regulated by virus–host interaction. Several viruses are known to use their late assembly (L) domains to hijack host machinery and cellular adaptors to be used for the requirement of virus replication. The L domains are highly conserved short sequences whose mutation or deletion may lead to the accumulation of immature virions at the plasma membrane. The L domains were firstly identified within retroviral Gag polyprotein and later detected in structural proteins of many other enveloped RNA viruses. Here, we used HIV-1 as an example to describe how the HIV-1 virus hijacks ESCRT membrane fission machinery to facilitate virion assembly and release. We also introduce galectin-3, a chimera type of the galectin family that is up-regulated by HIV-1 during infection and further used to promote HIV-1 assembly and budding via the stabilization of Alix–Gag interaction. It is worth further dissecting the details and finetuning the regulatory mechanism, as well as identifying novel candidates involved in this final step of replication cycle.