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: 5 × 10 3 cells/well of MDCK-TEVp or LAMP2A-KO MDCK cells cultured in 96-well plates were infected with two-fold serially diluted supernatants for 48 h. Then the cells were washed with PBS, fixed with 4% paraformaldehyde (PFA), washed with PBS, permeabilized with PBS containing 0.1% Triton X-100 (PBST) for 5 min, blocked with PBST containing 10% goat serum for 1 h at room temperature, and incubated with anti-M1 antibody (Sino Biological, Cat# 40010-RP01; 1:400 dilution) diluted in PBST containing 1% goat serum overnight at 4 °C and Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies, Cat# A11034; 1:1000 dilution) for 1 h at room temperature.

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: 5 × 10 3 cells/well of MDCK-TEVp or LAMP2A-KO MDCK cells cultured in 96-well plates were infected with two-fold serially diluted supernatants for 48 h. Then the cells were washed with PBS, fixed with 4% paraformaldehyde (PFA), washed with PBS, permeabilized with PBS containing 0.1% Triton X-100 (PBST) for 5 min, blocked with PBST containing 10% goat serum for 1 h at room temperature, and incubated with anti-M1 antibody (Sino Biological, Cat# 40010-RP01; 1:400 dilution) diluted in PBST containing 1% goat serum overnight at 4 °C and Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies, Cat# A11034; 1:1000 dilution) for 1 h at room temperature.

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

Journal: Advanced Science

Article Title: Attenuation of Influenza a Virus into Live Vaccines Through C‐End Degrons

doi: 10.1002/advs.202509425

Figure Lengend Snippet: Evaluation of the immunogenicity of M1 C−degron−1 , M1 C−degron−2 , and M1 C−degron−3 vaccines in C57BL/6J mice. A‐D, Serum antibody responses on day 21 after vaccination with 10 5 TCID 50 of the indicated vaccines (n = 5): NT (A), HI (B), anti‐HA IgG (C), and anti‐NP IgG (D). E, Virus‐specific IgA antibody responses in mouse lungs on day 21 after vaccination with 10 5 TCID 5 ₀ of the indicated vaccines (n = 5). F, Viral M1‐specific T cell responses in mouse lungs on day 7 after vaccination with 10 5 TCID 5 ₀ of the indicated vaccines (n = 5). IFN‐γ‐expressing cells per million cells are shown. G, Viral NP‐specific T cell responses in mouse lungs (left) and spleens (right) on day 7 after vaccination with 10 5 TCID 5 ₀ of the indicated vaccines (n = 5). IFN‐γ‐expressing cells per million cells are shown. H, PTD‐mediated enhancement of M1 antigen presentation in Raw264.7 cells. Cells were infected with M1 C−degron viruses (MOI = 1), and surface presentation of the M1 128‐135 epitope was detected 6 h post‐infection using an anti‐M1 peptide (M1 128‐135 ; MGLIYNRM) antibody. Data are expressed as mean ± s.d.; one‐way ANOVA with Tukey's multiple‐comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Primary antibodies included anti‐M1 (Sino Biological, 40010‐RP01, 1:1000 dilution) and anti‐β‐tubulin (Zen‐Bio, 700608, 1:10 000 dilution).

Techniques: Immunopeptidomics, Vaccines, Virus, Expressing, Infection