Journal: Nature Communications

Article Title: Lysosome-targeting live attenuated influenza vaccines elicit robust and broad immunity in mice

doi: 10.1038/s41467-026-69920-0

Figure Lengend Snippet: a LTM-dependent degradation of viral NS1 protein. Western blot analysis shows reduced NS1 protein levels in cells expressing NS1-N LTM compared with the N LTM mutant , while NS1 mRNA levels remain comparable ( n = 3). b Lysosome dependence of LTM-mediated NS1 degradation. NS1-N LTM protein degradation is blocked by lysosomal inhibitors but not by proteasome or autophagy inhibition. HEK293T cells expressing either NS1-N LTM (left) or NS1-N LTM mutant (right) protein were cultured with or without the proteasome inhibitor MG-132 (10 µM), the autophagy inhibitor 3-methyladenine (3-MA; 10 mM), bafilomycin A1 (Baf A1; 0.4 µM), or chloroquine (CQ; 50 µM) for 6 h. The viral NS1 protein was detected by Western blotting ( n = 3). c Co-immunoprecipitation demonstrating interaction of HSC70 with NS1-N LTM but not with the NS1 LTM mutant ( n = 3). d Dependence of LTM-mediated NS1 degradation on LAMP2A. Conventional HEK293T cells and LAMP2A-KO HEK293T cells were transfected with constructs expressing NS1-N LTM (left) or NS1-N LTM mutant (right) protein and collected 24 h post-transfection. Viral NS1 protein was detected by Western blotting ( n = 3). Immunofluorescence analysis showing colocalization of NS1-N LTM with HSC70 ( e ) and LAMP2A ( f ), but not of the NS1-N LTM mutant . Green, NS1; red, HSC70 or LAMP2A; blue, nuclei; yellow, colocalization sites; scale bar, 5 µm. Representative images of at least three independent experiments are shown. LAMP2A-dependent degradation of NS1-N LTM during viral infection in conventional and LAMP2A-KO HEK293T ( g ) and A549 ( h ) cells. Replication competence of NS1-N LTM and NS1-N LTM mutant viruses in conventional and LAMP2A-KO HEK293T ( i ) and A549 ( j ) cells. Immunofluorescence staining of influenza viral M1 protein at 48 h after infection (MOI = 0.01) showing the replication competence of NS1-N LTM or NS1-N LTM mutant virus in conventional and LAMP2A-KO cells. Green, M1; blue, nuclei; scale bar, 100 µm. Viral titers in culture supernatants were quantified by immunofluorescence focus-forming unit (FFU) assay ( n = 3). Data are means ± s.d; n = 3 biologically independent experiments; unpaired two-tailed t -test for ( i ) and ( j ); *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: The primary antibodies were anti-NS1 antibody (GeneTex, Cat# GTX125990, 1:2000 dilution), anti-PA antibody (GeneTex, Cat# GTX118991, 1:2000 dilution), anti-NP antibody (GeneTex, Cat# GTX125989, 1:5000 dilution), anti-M1 antibody (Sino Biological, Cat# 40010-RP01, 1:1000 dilution), anti-LAMP2A (Santa Cruz, Cat# sc-18822, 1:1000 dilution), and anti-β-actin (Proteintech Group, Cat# HRP-60008, 1:6000 dilution).

Techniques: Western Blot, Expressing, Mutagenesis, Inhibition, Cell Culture, Immunoprecipitation, Transfection, Construct, Immunofluorescence, Infection, Staining, Virus, Two Tailed Test

Journal: Nature Communications

Article Title: Lysosome-targeting live attenuated influenza vaccines elicit robust and broad immunity in mice

doi: 10.1038/s41467-026-69920-0

Figure Lengend Snippet: a Multi-cycle replication kinetics of the indicated viruses in conventional and LAMP2A-KO MDCK cells. Data are presented as means ± s.d ( n = 3). b triLTM-dependent degradation of viral proteins. Western blot analysis shows reduced levels of triLTM-tagged viral proteins compared with mutated triLTM-tagged controls, while corresponding mRNA levels remain comparable ( n = 3). c Lysosome dependence of triLTM-mediated viral protein degradation. HEK293T cells expressing triLTM-tagged or mutated triLTM-tagged viral proteins were cultured in the presence or absence of Baf A1 (0.4 µM) for 6 h and collected for detection of indicated viral proteins by Western blotting ( n = 3). d Co-immunoprecipitation demonstrating interaction of HSC70 with triLTM-tagged viral proteins but not with mutated triLTM-tagged viral proteins ( n = 3). LAMP2A-dependent degradation of triLTM-tagged viral proteins during viral infection in conventional and LAMP2A-KO HEK293T ( e ) and A549 ( f ) cells ( n = 3). Conventional cells and LAMP2A-KO cells were infected with LYTAR 2.0 dual triLTMs or LYTAR 2.0 dual triLTMs mutant virus and collected at 48 h after infection for detection of indicated proteins by Western blotting ( n = 3). Replication competence of LYTAR 2.0 dual triLTMs and LYTAR 2.0 dual triLTMs mutant viruses in conventional and LAMP2A-KO HEK293T ( g ) and A549 ( h ) cells. Immunofluorescence staining of influenza viral M1 protein at 48 h after infection (MOI = 0.01) showing the replication competence of LYTAR 2.0 dual triLTMs and LYTAR 2.0 dual triLTMs mutant virus in conventional and LAMP2A-KO HEK293T cells. Green, M1; blue, nuclei; scale bar, 100 µm. Viral titers in culture supernatants were quantified by immunofluorescence focus-forming unit (FFU) assay ( n = 3). Data are means ± s.d; n = 3 biologically independent experiments; unpaired two-tailed t -test for ( g ) and ( h ); *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: The primary antibodies were anti-NS1 antibody (GeneTex, Cat# GTX125990, 1:2000 dilution), anti-PA antibody (GeneTex, Cat# GTX118991, 1:2000 dilution), anti-NP antibody (GeneTex, Cat# GTX125989, 1:5000 dilution), anti-M1 antibody (Sino Biological, Cat# 40010-RP01, 1:1000 dilution), anti-LAMP2A (Santa Cruz, Cat# sc-18822, 1:1000 dilution), and anti-β-actin (Proteintech Group, Cat# HRP-60008, 1:6000 dilution).

Techniques: Western Blot, Expressing, Cell Culture, Immunoprecipitation, Infection, Mutagenesis, Virus, Immunofluorescence, Staining, Two Tailed Test

Journal: NPJ Vaccines

Article Title: Systemic prime mucosal boost significantly increases protective efficacy of bivalent RSV influenza viral vectored vaccine

doi: 10.1038/s41541-024-00912-1

Figure Lengend Snippet: a Vaccination and challenge schematic for the assessment of the protective capacity of ChAdOx1-NP + M1-RSVF against X31 (H3N2) infection and subsequent disease in mice. Mice were prime-boost-vaccinated, then challenged with X31, and culled 6 days later with tissues and fluids harvested. Blood sampling was performed 4 weeks post-prime and 3 weeks post-boost. b Weight change in mice over time post-challenge, measured as % of pre-challenge weight. Significant differences at timepoints between IM-IN and control mouse groups are represented with *, between IM-IN and IM-IM as # and between IN-IN and unvaccinated as @ (*, @ or # = p < 0.05, **= p < 0.01). c Viral load in lungs 6 days post-challenge (M gene copies/μg lung RNA (log 10 )). d H3N2 NP-specific IgG and IgA levels in serum, NWs, BALF and LHS post-challenge, as measured by ELISA (log 10 EU). Median negative control values are displayed as dashed lines. e Levels of antigen-specific CD8 + T RM cells, and relative levels of antigen-specific CD8 + T EM and T RM , in BAL and lungs post-challenge. T RM and T EM cells were defined as CD3 + CD8 + CD44 + CD62L - CD103 + CD69 + , and CD3 + CD8 + CD44 + CD62L - , respectively, and positive for influenza pentamer H-2Kd TYQRTALV. In boxplots, a “+” symbol represents the group mean. One mouse in group IM-IN did not have detectable Lung T RM . Group differences for all data were analysed using non-parametric Kruskal-Wallis tests (*= p < 0.05, **= p < 0.01). For all boxplots, whisker endings represent upper and lower extremes, the box bounds represent upper and lower quartiles, respectively, and the central line represents the group median.

Article Snippet: 96 well Nunc TM MaxiSorp TM plates were separately coated with 50μL/well of 2μg/mL (for tIgG and IgG subclass detection) or 5μg/mL (for IgA and IgM detection) recombinant Influenza A H1N1 (A/Puerto Rico/8/34/Mount Sinai) Nucleoprotein/NP I116M (ECD, His Tag), Influenza A H1N1 (A/Puerto Rico/8/34/Mount Sinai) Matrix protein 1 / M1 Protein (His Tag), Influenza A H3N2 (A/Hong Kong/2671/2019) Nucleoprotein / NP Protein (His Tag) Influenza A H3N2 (A/Aichi/2/1968) Matrix protein 1 / M1 Protein (His Tag) or Human respiratory syncytial virus (RSV) (A2) Fusion glycoprotein/RSV-F protein (ECD, His Tag) (all SinoBiological), overnight at 4 °C.

Techniques: Infection, Sampling, Control, Enzyme-linked Immunosorbent Assay, Negative Control, Whisker Assay