PUBLICATIONS

Monkeypox virus (Mpox) has been recognized for causing distinct skin lesions and is primarily transmitted through skin and sexual contact. To date, the transmissibility and pathogenesis of the Mpox virus in distal human lung has never been completely explored. Here the transmission pathways and Mpox tropism on patient-derived air-liquid epithelium (ALE) model fabricated using isolated primary human alveolar epithelial cells (hAECs) were investigated. hAECs were cultured and exposed to the Mpox virus clade IIb isolated from patient. DNA, proteins, and the tropism were elucidated using polymerase chain reaction (PCR), Western blot and high-content fluorescent imaging. Transmission electron microscopy (TEM) was employed to systematically observe the cellular distribution of viral particles. Viral titers were determined by TCID50 assay. Innate immune response and inflammatory mediators were measured using Milliplex® multiplex and ELISA analysis. Pathology at alveolar barrier integrity was determined using transepithelial electrical resistance (TEER) analysis. The study included mock-infected cells as control. Mpox virus significantly infected 42.82% of total hAEC populations. The prominent observed pathology included a significant reduction in TEER values, loss of tight junction protein, presence of tunneling nanotubes (TNTs) and syncytium morphology. Four stages of Mpox biogenesis were clearly observed without significant activation of IL-6, MIP1alpha, TNF-α, and Galectin-9, although IL-1β were subtly promoted. The developed patient-derived ALE is a versatile model for Mpox virus clade IIb infection reflecting respiratory transmission competence of the Mpox. Postinfection lung pathogenesis demonstrated alveolar barrier damage without significant inflammation, raising concerns about possible immune evasion by the virus.

In our study, Prodrug nucleoTide (ProTide) technology was applied to cordycepin to enhance its antiviral activities and metabolic stability. Using cordycepin as starting material, we developed a synthetic method to access a series of stereospecific-phosphoramidate derivatives with various ester groups. We also successfully synthesized halogenated cordycepin derivatives via stannylation. Our 17 ProTide-cordycepin derivatives were pharmacologically evaluated for their antiviral activities. Phosphorus diastereomers 22 S and 22 R showed moderate inhibitory activity against corona and influenza viruses, while these compounds and derivatives (25 S, 27 S, and 27 R) demonstrated promising antiviral efficacy against dengue virus. Pharmacological screening indicated that Sp-isomers generally exhibited slightly greater inhibitory activity than their Rp-isomer counterparts against the dengue virus. The selected ProTides were assessed for their metabolic mechanism and stability via carboxypeptidase and microsomes. The hydrolysis rate of the Rp-isomers was observed to be slightly higher than that of the Sp-isomers, and the addition of a fluorine group also modestly increased this rate and fluorinated 39 S extended its half-life compared to nonfluorinated counterparts. These findings suggested not only structure-activity relationships of cordycepin ProTide but also the comprehensive synthetic route to access cordycepin derivatives for further antiviral development.

Establishing a drug-screening platform is critical for the discovery of potential antiviral agents against SARS-CoV-2. In this study, we developed a platform based on human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to investigate SARS-CoV-2 infectivity, with the aim of evaluating potential antiviral agents for anti-SARS-CoV-2 activity and cardiotoxicity. Cultured myocytes of iPSC-CMs and immortalized human cardiomyocyte cell line (AC-16) were primarily characterized for the expression of cardiac markers and host receptors of SARS-CoV-2. An infectivity model for the wild-type SARS-CoV-2 strain was then established. Infection modeling involved inoculating cells with SARS-CoV-2 at varying multiplicities of infection (MOIs) and then quantifying infection using immunofluorescence and plaque assays. Only iPSC-CMs, not AC16 cells, expressed angiotensin-converting enzyme 2 (ACE-2), and quantitative assays confirmed the dose-dependent infection of iPSC-CMs by SARS-CoV-2, unlike the uninfectable AC16 cells lacking the expression of ACE2. Cytotoxicity was evaluated using MTT assays across a concentration range. An assessment of the plant-derived compound panduratin A (panA) showed cytotoxicity at higher doses (50% cytotoxic concentration (CC50) 10.09 μM) but promising antiviral activity against SARS-CoV-2 (50% inhibition concentration (IC50) 0.8-1.6 μM), suppressing infection at concentrations 10 times lower than its CC50. Plaque assays also showed decreased viral production following panA treatment. Overall, by modeling cardiac-specific infectivity, this iPSC-cardiomyocyte platform enables the reliable quantitative screening of compound cytotoxicity alongside antiviral efficacy. By combining disease pathogenesis and pharmacology, this system can facilitate the evaluation of potential novel therapeutics, such as panA, for drug discovery applications.

The ongoing COVID-19 pandemic has triggered extensive research, mainly focused on identifying effective therapeutic agents, specifically those targeting highly pathogenic SARS-CoV-2 variants. This study aimed to investigate the in vivo antiviral efficacy and anti-inflammatory activity of herbal extracts derived from Andrographis paniculata and Boesenbergia rotunda, using a Golden Syrian hamster model infected with Delta, a representative variant associated with severe COVID-19. Hamsters were intranasally inoculated with the SARS-CoV-2 Delta variant and orally administered either vehicle control, B. rotunda, or A. paniculata extract at a dosage of 1000 mg/kg/day. Euthanasia was conducted on days 1, 3, and 7 post-inoculation, with 4 animals per group. The results demonstrated that oral administration of A. paniculata extract significantly alleviated both lethality and infection severity compared with the vehicle control and B. rotunda extract. However, neither extract exhibited direct antiviral activity in terms of reducing viral load in the lungs. Nonetheless, A. paniculata extract treatment significantly reduced IL-6 protein levels in the lung tissue (7278 ± 868.4 pg/g tissue) compared to the control (12,495 ± 1118 pg/g tissue), indicating there was a decrease in local inflammation. This finding is evidenced by the ability of A. paniculata extract to reduce histological lesions in the lungs of infected hamsters. Furthermore, both extracts significantly decreased IL-6 and IP-10 mRNA expression in peripheral blood mononuclear cells of infected hamsters compared to the control group, suggesting systemic anti-inflammatory effects occurred. In conclusion, A. paniculata extract's potential therapeutic application for SARS-CoV-2 arises from its observed capacity to lessen inflammatory cytokine concentrations and mitigate lung pathology.

BACKGROUND: Andrographolide is a medicinal compound which possesses anti-SARS-CoV-2 activity. A number of cellular targets of andrographolide have been identified by target predictions and computational studies.

PURPOSE: However, a potential cellular target of andrographolide has never been explored in SARS-CoV-2 infected lung epithelial cells. We aimed to identify cellular pathways involved in andrographolide-mediated anti-SARS-CoV-2 activity.

METHODS: The viral infection was determined by immunofluorescence staining, enzyme-linked immunosorbent assay and focus-forming assay. Proteomic analysis was employed to identify cellular pathways and key proteins controlled by andrographolide in the human lung epithelial cells Calu-3 infected by SARS-CoV-2. Immunofluorescence staining was used to test protein expression and localization. Western blot and realtime PCR were utilized to elucidate gene expression. Cellular glutathione level was examined by a reduced/oxidized glutathione assay. An ectopic gene expression was delivered by plasmid transfection.

RESULTS: Gene ontology analysis indicates that proteins involved in nuclear factor erythroid 2-related factor 2 (NRF2)-regulated pathways were differentially expressed by andrographolide. Notably, andrographolide increased expression and nuclear localization of the transcription factor NRF2. In addition, transcriptional expression of GCLC and glutamate-cysteine ligase modifier subunit (GCLM), which are NRF2 target genes, were induced by andrographolide. We further find that infection of SARS-CoV-2 resulted in a reduction of glutathione level in Calu-3; the effect that was rescued by andrographolide. Moreover, andrographolide also induced expression of the glutathione producing enzyme GCLC in SARS-CoV-2 infected lung epithelial cells. Importantly, an ectopic over-expression of GCLC or treatment of N-acetyl-L-cysteine in Calu-3 cells led to a decrease in SARS-CoV-2 infection.

CONCLUSION: Collectively, our findings suggest the interplay between GCLC-mediated glutathione biogenesis induced by andrographolide and the anti-SARS-CoV-2 activity. The glutathione biogenesis and recycling pathways should be further exploited as a targeted therapy against SARS-CoV-2 infection.

Plant-based manufacturing has the advantage of post-translational modifications. While plant-specific N-glycans have been associated with allergic reactions, their effect on the specific immune response upon vaccination is not yet understood. In this study, we produced an RBD-Fc subunit vaccine in both wildtype (WT) and glycoengineered (∆XF) Nicotiana benthamiana plants. The N-glycan analysis: RBD-Fc carrying the ER retention peptide mainly displayed high mannose. When produced in WT RBD-Fc displayed complex-type (GnGnXF) N-glycans. In contrast, ∆XF plants produced RBD-Fc with humanized complex N-glycans that lack potentially immunogenic xylose and core fucose residues (GnGn). The three recombinant RBD-Fc glycovariants were tested. Immunization with any of the RBD-Fc proteins resulted in a similar titer of anti-RBD antibodies in mice. Likewise, antisera from subunit RBD-Fc vaccines also demonstrated comparable neutralization against SARS-CoV-2. Thus, we conclude that N-glycan modifications of the RBD-Fc protein have no impact on their capacity to activate immune responses and induce neutralizing antibody production.

BACKGROUND: Inactivated whole-virus vaccination elicits immune responses to both SARS-CoV-2 nucleocapsid (N) and spike (S) proteins, like natural infections. A heterologous Ad26.COV2.S booster given at two different intervals after primary BBIBP-CorV vaccination was safe and immunogenic at days 28 and 84, with higher immune responses observed after the longer pre-boost interval. We describe booster-specific and hybrid immune responses over 1 year.

METHODS: This open-label phase 1/2 study was conducted in healthy Thai adults aged ≥ 18 years who had completed primary BBIBP-CorV primary vaccination between 90-240 (Arm A1; n = 361) or 45-75 days (Arm A2; n = 104) before enrolment. All received an Ad26.COV2.S booster. We measured anti-S and anti-N IgG antibodies by Elecsys®, neutralizing antibodies by SARS-CoV-2 pseudovirus neutralization assay, and T-cell responses by quantitative interferon (IFN)-γ release assay. Immune responses were evaluated in the baseline-seronegative population (pre-booster anti-N < 1.4 U/mL; n = 241) that included the booster-effect subgroup (anti-N < 1.4 U/mL at each visit) and the hybrid-immunity subgroup (anti-N ≥ 1.4 U/mL and/or SARS-CoV-2 infection, irrespective of receiving non-study COVID-19 boosters).

RESULTS: In Arm A1 of the booster-effect subgroup, anti-S GMCs were 131-fold higher than baseline at day 336; neutralizing responses against ancestral SARS-CoV-2 were 5-fold higher than baseline at day 168; 4-fold against Omicron BA.2 at day 84. IFN-γ remained approximately 4-fold higher than baseline at days 168 and 336 in 18-59-year-olds. Booster-specific responses trended lower in Arm A2. In the hybrid-immunity subgroup at day 336, anti-S GMCs in A1 were 517-fold higher than baseline; neutralizing responses against ancestral SARS-CoV-2 and Omicron BA.2 were 28- and 31-fold higher, respectively, and IFN-γ was approximately 14-fold higher in 18-59-year-olds at day 336. Durable immune responses trended lower in ≥ 60-year-olds.

CONCLUSION: A heterologous Ad26.COV2.S booster after primary BBIBP-CorV vaccination induced booster-specific immune responses detectable up to 1 year that were higher in participants with hybrid immunity.

CLINICAL TRIALS REGISTRATION: NCT05109559.

ChulaCov19 mRNA vaccine demonstrated promising phase 1 results. Healthy adults aged 18-59 years were double-blind randomised 4:1 to receive two intramuscular doses of ChulaCov19 50 µg or placebo. Primary endpoints were safety and microneutralization antibody against-wild-type (Micro-VNT50) at day 50. One hundred fifty adults with median (IQR) age 37 (30-46) years were randomised. ChulaCov19 was well tolerated, and most adverse events were mild to moderate and temporary. Geometric mean titres (GMT) of neutralizing titre against wild-type for ChulaCov19 on day 50 were 1367 IU/mL. T-cell IFN-γ-ELISpot showed the highest responses at one week (Day29) after dose 2 then gradually declined. ChulaCov19 50 µg is well tolerated and elicited high neutralizing antibodies and strong T-cell responses in healthy adults.Trial registration number: ClinicalTrials.gov Identifier NCT04566276, 28/09/2020.

Respiratory syncytial virus (RSV) is a highly contagious virus that affects the lungs and respiratory passages of many vulnerable people. It is a leading cause of lower respiratory tract infections and clinical complications, particularly among infants and elderly. It can develop into serious complications such as pneumonia and bronchiolitis. The development of RSV vaccine or immunoprophylaxis remains highly active and a global health priority. Currently, GSK's Arexvy™ vaccine is approved for the prevention of lower respiratory tract disease in older adults (>60 years). Palivizumab and currently nirsevimab are the approved monoclonal antibodies (mAbs) for RSV prevention in high-risk patients. Many studies are ongoing to develop additional therapeutic antibodies for preventing RSV infections among newborns and other susceptible groups. Recently, additional antibodies have been discovered and shown greater potential for development as therapeutic alternatives to palivizumab and nirsevimab. Plant expression platforms have proven successful in producing recombinant proteins, including antibodies, offering a potential cost-effective alternative to mammalian expression platforms. Hence in this study, an attempt was made to use a plant expression platform to produce two anti-RSV fusion (F) mAbs 5C4 and CR9501. The heavy-chain and light-chain sequences of both these antibodies were transiently expressed in Nicotiana benthamiana plants using a geminiviral vector and then purified using single-step protein A affinity column chromatography. Both these plant-produced mAbs showed specific binding to the RSV fusion protein and demonstrate effective viral neutralization activity in vitro. These preliminary findings suggest that plant-produced anti-RSV mAbs are able to neutralize RSV in vitro.