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In view of addressing the global necessity of an effective vaccine in the SARS-CoV-2 pandemic, a plasmid DNA vaccine, expressing for the spike (S) protein and formulated in lipoplexes, was manufactured and tested for in vitro transfection and in vivo immunogenicity. Blank cationic liposomes of 130.9 ± 5.8 nm in size and with a zeta potential of +48 ± 12 mV were formulated using the thin-film layer rehydration method. Liposomes were complexed with pCMVkan-S at different N/P ratios. Ratios of 0.25:1 and 1:1 were selected according to their complex stability and controlled size compared to other ratios and tested in vitro for transfection studies and in vivo for immunogenicity. Both selected formulations showed enhanced neutralizing antibody responses compared to pCMVkan-S injected alone, as well as an increased T cell response. The titers observed were similar to those of intramuscular electroporation (IM-EP), which was set as an efficacy goal.

Due to the rapid transmission of the coronavirus disease 2019 (COVID-19) causing serious public health problems and economic burden, the development of effective vaccines is a high priority for controlling the virus spread. Our group has previously demonstrated that the plant-produced receptor-binding domain (RBD) of SARS-CoV-2 fused with Fc of human IgG was capable of eliciting potent neutralizing antibody and cellular immune responses in animal studies, and the immunogenicity could be improved by the addition of an alum adjuvant. Here, we performed a head-to-head comparison of different commercially available adjuvants, including aluminum hydroxide gel (alum), AddaVax (MF59), monophosphoryl lipid A from Salmonella minnesota R595 (mPLA-SM), and polyinosinic-polycytidylic acid (poly(I:C)), in mice by combining them with plant-produced RBD-Fc, and the differences in the immunogenicity of RBD-Fc with different adjuvants were evaluated. The specific antibody responses in terms of total IgG, IgG1, and IgG2a subtypes and neutralizing antibodies, as well as vaccine-specific T-lymphocyte responses, induced by the different tested adjuvants were compared. We observed that all adjuvants tested here induced a high level of total IgG and neutralizing antibodies, but mPLA-SM and poly (I:C) showed the induction of a balanced IgG1 and IgG2a (Th2/Th1) immune response. Further, poly (I:C) significantly increased the frequency of IFN-γ-expressing cells compared with control, whereas no significant difference was observed between the adjuvanted groups. This data revealed the adjuvants' role in enhancing the immune response of RBD-Fc vaccination and the immune profiles elicited by different adjuvants, which could prove helpful for the rational development of next-generation SARS-CoV-2 RBD-Fc subunit vaccines. However, additional research is essential to further investigate the efficacy and safety of this vaccine formulation before clinical trials.

Updated and revised versions of COVID-19 vaccines are vital due to genetic variations of the SARS-CoV-2 spike antigen. Furthermore, vaccines that are safe, cost-effective, and logistic-friendly are critically needed for global equity, especially for middle- to low-income countries. Recombinant protein-based subunit vaccines against SARS-CoV-2 have been reported using the receptor-binding domain (RBD) and the prefusion spike trimers (S-2P). Recently, a new version of prefusion spike trimers, named HexaPro, has been shown to possess two RBD in the "up" conformation, due to its physical property, as opposed to just one exposed RBD found in S-2P. Importantly, this HexaPro spike antigen is more stable than S-2P, raising its feasibility for global logistics and supply chain. Here, we report that the spike protein HexaPro offers a promising candidate for the SARS-CoV-2 vaccine. Mice immunized by the recombinant HexaPro adjuvanted with aluminum hydroxide using a prime-boost regimen produced high-titer neutralizing antibodies for up to 56 days after initial immunization against live SARS-CoV-2 infection. Also, the level of neutralization activity is comparable to that of convalescence sera. Our results indicate that the HexaPro subunit vaccine confers neutralization activity in sera collected from mice receiving the prime-boost regimen.

The emergence of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected global public health and economy. Despite the substantial efforts, only few vaccines are currently approved and some are in the different stages of clinical trials. As the disease rapidly spreads, an affordable and effective vaccine is urgently needed. In this study, we investigated the immunogenicity of plant-produced receptor-binding domain (RBD) of SARS-CoV-2 in order to use as a subunit vaccine. In this regard, RBD of SARS-CoV-2 was fused with Fc fragment of human IgG1 and transiently expressed in Nicotiana benthamiana by agroinfiltration. The plant-produced RBD-Fc fusion protein was purified from the crude extract by using protein A affinity column chromatography. Two intramuscular administration of plant-produced RBD-Fc protein formulated with alum as an adjuvant have elicited high neutralization titers in immunized mice and cynomolgus monkeys. Further it has induced a mixed Th1/Th2 immune responses and vaccine-specific T-lymphocyte responses which was confirmed by interferon-gamma (IFN-γ) enzyme-linked immunospot assay. Altogether, our results demonstrated that the plant-produced SARS-CoV-2 RBD has the potential to be used as an effective vaccine candidate against SARS-CoV-2. To our knowledge, this is the first report demonstrating the immunogenicity of plant-produced SARS-CoV-2 RBD protein in mice and non-human primates.

Viruses have developed direct cell-to-cell transfer strategies to enter target cells without being released to escape host immune responses and antiviral treatments. These strategies are more rapid and efficient than transmission through indirect mechanisms of viral infection between cells. Here, we demonstrate that an H5N1 influenza virus can spread via direct cell-to-cell transfer in Madin-Darby canine kidney (MDCK) cells. We compared cell-to-cell transmission of the H5N1 virus to that of a human influenza H1N1 virus. The H5N1 virus has been found to spread to recipient cells faster than the human influenza H1N1 virus. Additionally, we showed that plasma membrane exchange (trogocytosis) occurs between co-cultured infected donor cells and uninfected recipient cells early point, allowing the intercellular transfer of viral material to recipient cells. Notably, the H5N1 virus induced higher trogocytosis levels than the H1N1 virus, which could explain the faster cell-to-cell transmission rate of H5N1. Importantly, this phenomenon was also observed in A549 human lung epithelial cells, which are representative cells in the natural infection site. Altogether, our results provide evidence demonstrating that trogocytosis could be the additional mechanism utilized by the H5N1 virus for rapid and efficient cell-to-cell transmission.

Despite being an important health problem, there are only supportive care treatments for respiratory syncytial virus (RSV) infection. Thus, discovery of specific therapeutic drugs for RSV is still needed. Recently, an antiparasitic drug niclosamide has shown a broad-spectrum antiviral activity. Here, our in vitro model was used to study the antiviral effect of niclosamide on RSV and its related mechanism. Niclosamide inhibited RSV with time and dose-dependent manner. Pretreatment with submicromolar concentration of niclosamide for 6 h presented the highest anti-RSV activity of 94 % (50 % effective concentration; EC₅₀ of 0.022 μM). Niclosamide efficiently blocked infection of laboratory strains and clinical isolates of both RSV-A and RSV-B in a bronchial epithelial cell line. Although a disruption of the mechanistic target of rapamycin complex 1 (mTORC1) pathway by niclosamide was previously hypothesized as a mechanism against pH-independent viruses like RSV, using a chemical mTORC1 inhibitor, temsirolimus, and a chemical mTORC1 agonist, MHY1485 (MHY), we show here that the mechanism of RSV inhibition by niclosamide was mTORC1 independent. Indeed, our data indicated that niclosamide hindered RSV infection via proapoptotic activity by a reduction of AKT prosurvival protein, activation of cleaved caspase-3 and PARP (poly ADP-ribose polymerase), and an early apoptosis induction.

The coronaviruses disease 2019 (COVID-19) caused by a novel coronavirus (SARS-CoV-2) has become a major health problem, affecting more than 50 million people with over one million deaths globally. Effective antivirals are still lacking. Here, we optimized a high-content imaging platform and the plaque assay for viral output study using the legitimate model of human lung epithelial cells, Calu-3, to determine the anti-SARS-CoV-2 activity of Andrographis paniculata extract and its major component, andrographolide. SARS-CoV-2 at 25TCID₅₀ was able to reach the maximal infectivity of 95% in Calu-3 cells. Postinfection treatment of A. paniculata and andrographolide in SARS-CoV-2-infected Calu-3 cells significantly inhibited the production of infectious virions with an IC₅₀ of 0.036 μg/mL and 0.034 μM, respectively, as determined by the plaque assay. The cytotoxicity profile developed over the cell line representatives of major organs, including liver (HepG2 and imHC), kidney (HK-2), intestine (Caco-2), lung (Calu-3), and brain (SH-SY5Y), showed a CC₅₀ of >100 μg/mL for A. paniculata extract and 13.2–81.5 μM for andrographolide, respectively, corresponding to a selectivity index of over 380. In conclusion, this study provided experimental evidence in favor of A. paniculata and andrographolide for further development as a monotherapy or in combination with other effective drugs against SARS-CoV-2 infection.

DC-SIGN, a C-type lectin mainly expressed in dendritic cells (DCs), has been reported to mediate several viral infections. We previously reported that DC-SIGN mediated H5N1 influenza A virus (AIVs) infection, however, the important DC-SIGN interaction with N-glycosylation sites remain unknown. This study aims to identify the optimal DC-SIGN interacting N-glycosylation sites in HA proteins of H5N1-AIVs. Results from NetNGlyc program analyzed the H5 hemagglutinin sequences of isolates during 2004-2020, revealing that seven and two conserved N-glycosylation sites were detected in HA1 and HA2 domain, respectively. A lentivirus pseudotyped A/Vietnam/1203/04 H5N1 envelope (H5N1-PVs) was generated which displayed an abundance of HA5 proteins on the virions via immuno-electron microscope observation. Further, H5N1-PVs or reverse-genetics (H5N1-RG) strains carrying a serial N-glycosylated mutation was generated by site-directed mutagenesis assay. Human recombinant DC-SIGN (rDC-SIGN) coated ELISA showed that H5N1-PVs bound to DC-SIGN, however, mutation on the N27Q, N39Q, and N181Q significantly reduced this binding (p < 0.05). Infectivity and capture assay demonstrated that N27Q and N39Q mutations significantly ameliorated DC-SIGN mediated H5N1 infection. Furthermore, combined mutations (N27Q&N39Q) significantly waned the interaction on either H5N1-PVs or -RG infection in cis and in trans (p < 0.01). This study concludes that N27 and N39 are two essential N-glycosylation contributing to DC-SIGN mediating H5N1 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease (COVID-19) which has recently emerged as a potential threat to global public health. SARS-CoV-2 is the third known human coronavirus that has huge impact on the human population after SARS-CoV and MERS-CoV. Although some vaccines and therapeutic drugs are currently in clinical trials, none of them are approved for commercial use yet. As with SARS-CoV, SARS-CoV-2 utilizes angiotensin-converting enzyme 2 (ACE2) as the cell entry receptor to enter into the host cell. In this study, we have transiently produced human ACE2 fused with the Fc region of human IgG1 in Nicotiana benthamiana and the in vitro neutralization efficacy of the plant-produced ACE2-Fc fusion protein was assessed. The recombinant ACE2-Fc fusion protein was expressed in N. benthamiana at 100 μg/g leaf fresh weight on day 6 post-infiltration. The recombinant fusion protein showed potent binding to receptor binding domain (RBD) of SARS-CoV-2. Importantly, the plant-produced fusion protein exhibited potent anti-SARS-CoV-2 activity in vitro. Treatment with ACE2-Fc fusion protein after viral infection dramatically inhibit SARS-CoV-2 infectivity in Vero cells with an IC₅₀ value of 0.84 μg/ml. Moreover, treatment with ACE2-Fc fusion protein at the pre-entry stage suppressed SARS-CoV-2 infection with an IC₅₀ of 94.66 μg/ml. These findings put a spotlight on the plant-produced ACE2-Fc fusion protein as a potential therapeutic candidate against SARS-CoV-2.