Rapid and Efficient Cell-to-Cell Transmission of Avian Influenza H5N1 Virus in MDCK Cells Is Achieved by Trogocytosis

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

Immunogenicity and efficacy of recombinant subunit SARS-CoV-2 vaccine candidate in the Syrian hamster model

SARS-CoV-2 causes devastating impact on the human population and has become a major public health concern. The frequent emergence of SARS-CoV-2 variants of concern urges the development of safe and efficacious vaccine against SARS-CoV-2 variants. We developed a candidate vaccine Baiya SARS-CoV-2 Vax 1, based on SARS-CoV-2 receptor-binding domain (RBD) by fusing with the Fc region of human IgG. The RBD-Fc fusion was produced in Nicotiana benthamiana. Previously, we reported that this plant-produced vaccine is effective in inducing immune response in both mice and non-human primates. Here, the efficacy of our vaccine candidate was tested in Syrian hamster challenge model. Hamsters immunized with two intramuscular doses of Baiya SARS-CoV-2 Vax 1 induced neutralizing antibodies against SARS-CoV-2 and protected from SARS-CoV-2 challenge with reduced viral load in the lungs. These preliminary results demonstrate the ability of plant-produced subunit vaccine Baiya SARS-CoV-2 Vax 1 to provide protection against SARS-CoV-2 infection in hamsters

Trogocytosis with monocytes associated with increased α2,3 sialic acid expression on B cells during H5N1 influenza virus infection.

The immunopathogenesis of H5N1 virus has been studied intensively since it caused cross-species infection and induced high mortality to human. We previously observed the interaction between monocytes and B cells, which increased the susceptibility of B cell to H5N1 virus infection after a co-culture. Levels of α2,3 sialic acid (avian flu receptor) were also significantly increased on B cell surface in this co-culture model with unclear explanation. In this study, we aimed to determine the possible mechanism that responded for this increase in α2,3 sialic acid on B cells. Acquisition of α2,3 SA by B cells via cell contact-dependent trogocytosis was proposed. Results showed that the lack of α2,3 SA was detected on B cell surface, and B cells acquired membrane-bound α2,3 SA molecules from monocytes in H5N1-infected co-cultures. Occurrence of membrane exchange mainly relied on H5N1 infection and cell-cell contact as opposed to a mock infection and transwell. The increase in α2,3 SA on B cell surface mediated by trogocytosis was associated with the enhanced susceptibility to H5N1 infection. These observations thus provide the evidence that H5N1 influenza virus may utilize trogocytosis to expand its cell tropism and spread to immune cells despite the lack of avian flu receptor

Immunogenicity Studies of Plant-Produced SARS-CoV-2 Receptor Binding Domain-Based Subunit Vaccine Candidate with Different Adjuvant Formulations

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

Target Enrichment Metagenomics Reveals Human Pegivirus-1 in Pediatric Hematopoietic Stem Cell Transplantation Recipients

Human pegivirus-1 (HPgV-1) is a lymphotropic human virus, typically considered nonpathogenic, but its infection can sometimes cause persistent viremia both in immunocompetent and immunosuppressed individuals. In a viral discovery research program in hematopoietic stem cell transplant (HSCT) pediatric patients, HPgV-1 was detected in 3 out of 14 patients (21.4%) using a target enrichment next-generation sequencing method, and the presence of the viruses was confirmed by agent-specific qRT-PCR assays. For the first time in this patient cohort, complete genomes of HPgV-1 were acquired and characterized. Phylogenetic analyses indicated that two patients had HPgV-1 genotype 2 and one had HPgV-1 genotype 3. Intra-host genomic variations were described and discussed. Our results highlight the necessity to screen HSCT patients and blood and stem cell donors to reduce the potential risk of HPgV-1 transmission

DNA Vaccine Administered by Cationic Lipoplexes or by In Vivo Electroporation Induces Comparable Antibody Responses against SARS-CoV-2 in Mice

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.</p

Translating a Thin-Film Rehydration Method to Microfluidics for the Preparation of a SARS-CoV-2 DNA Vaccine: When Manufacturing Method Matters

Previous investigations conducted on a liposomal formulation for a SARS-CoV-2 DNA vaccine manufactured using the thin-film layer rehydration method showed promising immunogenicity results in mice. The adaptation of the liposomal formulation to a scalable and reproducible method of manufacture is necessary to continue the investigation of this vaccine candidate. Microfluidics manufacture shows high potential in method translation. The physicochemical characterization of the blank liposomes produced by thin-film layer rehydration or microfluidics were shown to be comparable. However, a difference in lipid nanostructure in the bilayer resulted in a significant difference in the hydration of the thin-film liposomes, ultimately altering their complexation behavior. A study on the complexation of liposomes with the DNA vaccine at various N/P ratios showed different sizes and Zeta-potential values between the two formulations. This difference in the complexation behavior resulted in distinct immunogenicity profiles in mice. The thin-film layer rehydration-manufactured liposomes induced a significantly higher response compared to the microfluidics-manufactured samples. The nanostructural analysis of the two samples revealed the critical importance of understanding the differences between the two formulations that resulted in the different immunogenicity in mice

Mice Immunized with the Vaccine Candidate HexaPro Spike Produce Neutralizing Antibodies against SARS-CoV-2

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