Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Neuraminidase delivered as an APC-targeted DNA vaccine induces protective antibodies against influenza.

doi: 10.1016/j.ymthe.2023.03.012

Figure Lengend Snippet: Figure 1. In vitro characterization of MHC class II-targeted heterodimeric DNA vaccines containing NA and/or HA (A) MHC class II-targeted heterodimeric acid/base (A/B) vaccine proteins consist of A and B chains, each having a targeting, dimerization, and antigenic unit. The targeting unit is either an scFv that binds MHC class II on APCs (scFvaMHCII, green) or a non-targeted control that binds the hapten NIP (scFvaNIP, white). The targeting units are genetically linked to a shortened hinge from human IgG3 (exon h1), allowing covalent disulfide bond formation between the A and B chains (black lines). The hinge is followed by a duplicated A (red) or B (blue) heterodimerization unit based on a modified Jun/Fos leucine zipper motif enriched for acidic or basic amino acids, respectively. The A and B chains are linked to antigenic units composed of NA (dark blue) or HA (orange) derived from influenza H1N1 PR8 virus. The heterodimeric vaccine proteins are named as

Article Snippet: The copy number of viral RNA was determined with quantitative genomic RNA from H1N1 PR8 (VR-95DQ, ATCC) and shown per microgram total tissue RNA.

Techniques: In Vitro, Vaccines, Control, Derivative Assay, Virus

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Neuraminidase delivered as an APC-targeted DNA vaccine induces protective antibodies against influenza.

doi: 10.1016/j.ymthe.2023.03.012

Figure Lengend Snippet: Figure 2. MHC class II-targeted NA-bivalent heterodimers efficiently induce antibody responses after a single DNA vaccination (A) BALB/c mice were vaccinated once i.m. with 5 mg of each A and B plasmids (10 mg DNA in total) encoding the indicated heterodimeric vaccine proteins (box), followed immediately by EP. Blood was sampled at the indicated time points. (B) NA-specific (top) and HA-specific (bottom) IgG (left), IgG1 (center), and IgG2a (right) titers were measured by ELISA at the indicated time points after immunization. Statistical analysis compared targeted vs. non-targeted and antigen-bivalent vs. monovalent groups. (C) BALB/c mice were immunized once i.m./EP with different amounts of the indicated DNA vaccines. Serum was analyzed for NA-specific IgG responses 4 weeks post vaccination. (D) IgG responses specific for inactivated H1N1 PR8 virus (left), rec. NA protein (center), or rec. HA protein (right) in sera harvested 6 weeks post vaccination from

Article Snippet: The copy number of viral RNA was determined with quantitative genomic RNA from H1N1 PR8 (VR-95DQ, ATCC) and shown per microgram total tissue RNA.

Techniques: Enzyme-linked Immunosorbent Assay, Vaccines, Virus

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Neuraminidase delivered as an APC-targeted DNA vaccine induces protective antibodies against influenza.

doi: 10.1016/j.ymthe.2023.03.012

Figure Lengend Snippet: Figure 4. A single DNA immunization with MHC class II-targeted NA/NA vaccines protects against homologous influenza virus challenge (A) BALB/c mice were immunized once i.m./EP and challenged with homologous influenza A H1N1 PR8 virus at week 2 (short term) or between weeks 6 and 10 (long term) after vaccination. (B–F) 50 mg of each A and B plasmids (total 100 mg, B), 5 mg/ plasmid (10 mg total, C–E), or 1 mg/plasmid (2 mg total, F) of the indicated DNA vaccines (box) were used. Mice were challenged with a lethal dose of 5 LD50 (B–D and F) or 100 LD50 (E) at week 9 (B), week 10 (C), week 2 (D and E), or week 6 (F). Weight (left, mean ± SEM) and survival (right) were monitored up to 12 days post challenge. n = 8 mice/group (B, C, E, and F) or n = 6 mice/group (D). Dead mice were assigned a weight of 75%. *p % 0.05, ***p % 0.001; two-way ANOVA (weight curve) or Mantel-Cox test (survival). Statistical analysis compared MHC class II- targeted NA/NA versus HA/HA as well as targeted versus non-targeted and anti- gen-bivalent versus -monovalent NA.

Article Snippet: The copy number of viral RNA was determined with quantitative genomic RNA from H1N1 PR8 (VR-95DQ, ATCC) and shown per microgram total tissue RNA.

Techniques: Vaccines, Virus, Plasmid Preparation

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Neuraminidase delivered as an APC-targeted DNA vaccine induces protective antibodies against influenza.

doi: 10.1016/j.ymthe.2023.03.012

Figure Lengend Snippet: Figure 5. MHC class II-targeted NA DNA vaccine induces superior NA-specific antibody responses compared with conventional inactivated virus after one vaccination (A, B) BALB/c mice were immunized once i.m. with 0.2, 1, or 5 mg of formalin-inactivated H1N1 PR8 virus or with 5 mg/ plasmid (10 mg total) of the indicated DNA vaccines, fol- lowed by EP (box). (A) NA-specific (left) and HA-specific (right) titers were measured by ELISA at the indicated time points after immunization. Mean ± SEM is shown. (B) Mice were challenged with 5 LD50 H1N1 PR8 virus 11 week post vaccination. Weight (left, mean ± SEM) and survival (right) were monitored up to 12 days post challenge. Dead mice were assigned a weight of 75%; n = 8 mice/group. Statistical analysis compared the different doses of inactivated virus with DNA vaccination with MHC class II-targeted NA and/or HA. **p % 0.01, ***p % 0.001; two-way ANOVA (IgG titers, weight curve) or Mantel-Cox test (survival).

Article Snippet: The copy number of viral RNA was determined with quantitative genomic RNA from H1N1 PR8 (VR-95DQ, ATCC) and shown per microgram total tissue RNA.

Techniques: Virus, Plasmid Preparation, Vaccines, Enzyme-linked Immunosorbent Assay

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Neuraminidase delivered as an APC-targeted DNA vaccine induces protective antibodies against influenza.

doi: 10.1016/j.ymthe.2023.03.012

Figure Lengend Snippet: Figure 6. Vaccine-induced NA-specific antibodies mediate protection against homologous influenza infection, inhibit NA enzymatic activity, and block viral replication (A–D) BALB/c mice were immunized once i.m./EP with 50 mg each of A and B plasmids (100 mg total) as indicated. (A) Pooled sera (350 mL) from mice 5 weeks after vaccination were transferred i.p. to naive BALB/c mice. One day after transfer, mice were challenged with 2.5 LD50 H1N1 PR8 virus. (B) 2 weeks after vaccination with the indicated DNA vaccines, mice were challenged with 5 LD50 H1N1 PR8 virus. Starting on day 12 after vaccination, mice were treated with anti-CD4 and anti- CD8 mAbs or isotype-matched control mAbs every second day. n = 8 mice/group; weight (left, mean ± SEM) and survival (right) were monitored up to 12 days post challenge, and dead mice were assigned a weight of 75% (A and B). (C) Neutrali- zation of influenza A H1N1 PR8 virus by serum from vaccinated mice (box) obtained 4 weeks after immunization was measured by microneutralization assay and is shown as IC50. (D) The same sera were tested for ability to inhibit NA enzyme activity by ELLA, using a reassorted influenza A H6N1 virus containing NA from H1N1 PR8. IC50 is indicated. (E and F) BALB/c mice were immunized once i.m./EP with 50 mg of each A and B plasmids (100 mg total, E) or 5 mg of each A and B plasmids (10 mg total, F) as indicated. Mice were challenged with homologous H1N1 PR8 virus 6 weeks after vaccination, and lungs were harvested on day 5 post challenge. Viral loads were determined by qRT-PCR (left), showing gene copy number per micro- gram total tissue RNA, and by measuring in vitro cell culture infectivity (right), pre- sented as 50% tissue culture infectious dose (TCID50) per milliliter of lung homog- enates. Individual mice and mean ± SEM are shown; n = 4–6 mice/group (C–F). *p % 0.05, **p % 0.01, ***p % 0.001; two-way ANOVA (weight curve), Mantel-Cox test (survival), Kruskal-Wallis multiple-comparisons test with Dunn’s correction (C and D, TCID50), or multiple-comparisons one-way ANOVA with Tukey’s correction (copy number).

Article Snippet: The copy number of viral RNA was determined with quantitative genomic RNA from H1N1 PR8 (VR-95DQ, ATCC) and shown per microgram total tissue RNA.

Techniques: Infection, Activity Assay, Blocking Assay, Virus, Vaccines, Control, Microneutralization Assay, Quantitative RT-PCR, In Vitro, Cell Culture

Journal: Nature medicine

Article Title: Programmable antivirals targeting critical conserved viral RNA secondary structures from influenza A virus and SARS-CoV-2

doi: 10.1038/s41591-022-01908-x

Figure Lengend Snippet: SHAPE-chemical mapping performed on full-length (−)-sense PB2 vRNAs. Colors denote SHAPE reactivity, which is proportional to the probability that a nucleotide is single-stranded. All structures are truncated to highlight the 5′ termini sequence structure. (a) SHAPE-predicted wild-type PB2 RNA secondary structure from strain A/Puerto Rico/8/1934 “PR8” (H1N1). Color-coded circles correspond to nucleotides sites where synonymous mutations were reported to affect PB2 packaging11,14. (b) Packaging efficiency of synonymous mutants in (a), determined by RT-qPCR. Results representative of two independent experiments with biological replicates, each performed in triplicate. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test against the WT mean by GraphPad Prism 9 software (n=4). Error bars represents mean ± standard deviation (s.d.); **** p < 0.0001; * p = 0.0454, n.s. = not significant. Box below indicates mutant name and corresponding nucleotide change. Nucleotide numbering shown in the genomic (−)-sense orientation. (c) SHAPE-mapped structures of PB2 packaging-defective mutant vRNAs, m757 (G44C) and m745 (A80U) indicating loss of PSL2’s RNA secondary structure. Black arrowheads and boxed nucleotides denote site(s) of synonymous mutation. (d) Web-logo representation of the PSL2 region conservation across IAV strains and diverse influenza A viral subtypes (weblogo.berkeley.edu). The overall height represents sequence conservation at that nucleotide position, while the symbol height within each position indicates the relative frequency of each nucleotide at that site. Black box denotes PSL2 region. Sequences included in the alignment: pandemic A/Brevig Mission/1/1918 (H1N1), pandemic A/California/04/2009 (H1N1), seasonal human A/New York/470/2004 (H3N2), A/Puerto Rico/8/1934 (H1N1), highly pathogenic avian A/Vietnam/03/2004 (H5N1), avian A/mallard/Maryland/14OS1154/2014 (H6N1), pandemic A/Hong Kong/8/1968 (H3N2), and seasonal human A/New York/312/2001 (H1N1) (see Supplementary Fig. 1d). RNA nucleotides are numbered in (−) -sense orientation. (e) SHAPE-mapped structures of full-length wild-type PB2 vRNA from pandemic and highly pathogenic strains, including different subtypes to modern human strains: 1918 pandemic (A/Brevig Mission/1/1918 (H1N1)), highly-pathogenic avian (A/Vietnam/1203/2004 (H5N1)), 2009 pandemic ‘swine’ (A/California/04/2009 (H1N1)), and Fujian-like human seasonal virus, A/New York/470/2004 (H3N2)

Article Snippet: Tissue-cultured adapted influenza A/Hong Kong/8/68 (HK68) H3N2 virus (ATCC-VR-1679), A/Virginia/ATCC6/2012 (H3N2) virus (ATCC-VR-1811), A/Virginia/ATCC1/2009TC (H1N1) virus (ATCC-VR-1736), and A/Wisconsin/33 (H1N1) virus (VR-1520) were purchased from ATCC.

Techniques: Sequencing, Quantitative RT-PCR, Software, Standard Deviation, Mutagenesis, Virus

Journal: Nature medicine

Article Title: Programmable antivirals targeting critical conserved viral RNA secondary structures from influenza A virus and SARS-CoV-2

doi: 10.1038/s41591-022-01908-x

Figure Lengend Snippet: (a) Regions of PSL2 targeted by indicated LNAs. (b) Antiviral screen of LNAs transfected into MDCK cells and infected 4 hours later with PR8 (H1N1) or A/Hong Kong/8/68 (H3N2) virus (0.01 MOI) and viral titers determined 48 hours post infection (n=3). Statistics performed by unpaired ordinary one-way ANOVA (PR8 and HK68) with Dunnett’s multiple comparisons. (c) Antiviral efficacy as a function of time of LNA addition (n=3), analyzed as in (b). Statistics by 2-way ANOVA with Dunnett’s multiple comparisons test against non-treated +Lipo3k (N.T.). (d) PB2 vRNA (PR8) packaging efficiency of viruses treated with 100 nM LNA9 or Scr. LNA control. Values given as a percentage of PB2 vRNA packaging in comparison to non-treated wild-type PR8 virus; readout by qPCR. Results from two biological replicates (n=2), assays performed in technical triplicates. (n=6). (e) LNA9 efficacy against multiple IAV strains in MDCK cells pretreated with 100 nM of indicated LNAs. Analyzed as in (b). Statistics as described in (c) against the N.T. control. (f) In vitro selection for drug resistance to LNA9 with escalating concentrations of LNA and the sensitivity of passaged virus in response to drug treatment. EC50s determined at the indicated passage (P) numbers. Results expressed as a percentage of nontreated virus titer. (g) In vitro selection of PR8 virus selected with oseltamivir carboxylate (OSLT). OSLT-treated PR8 virus and drug sensitivity determined by plaque reduction assay. The number of viral plaques with each drug concentration was normalized against the nontreated control to determine the EC50. (h, i) In vitro sensitivity of wild-type WSN33 (H1N1) and neuraminidase (NA) inhibitor-resistant (WSN H275Y NA mutant) virus to LNA9 (h) or OSLT (i). EC50 values were computed using a nonlinear regression model with variable slope. Statistics for all graphs performed in GraphPad Prism 9 software. All error bars represent mean ± s.d. * Statistical P value as indicated in each panel.

Article Snippet: Tissue-cultured adapted influenza A/Hong Kong/8/68 (HK68) H3N2 virus (ATCC-VR-1679), A/Virginia/ATCC6/2012 (H3N2) virus (ATCC-VR-1811), A/Virginia/ATCC1/2009TC (H1N1) virus (ATCC-VR-1736), and A/Wisconsin/33 (H1N1) virus (VR-1520) were purchased from ATCC.

Techniques: Transfection, Infection, Virus, Control, Comparison, In Vitro, Selection, Concentration Assay, Mutagenesis, Software

Journal: PLoS ONE

Article Title: Development of an efficient Sanger sequencing-based assay for detecting SARS-CoV-2 spike mutations

doi: 10.1371/journal.pone.0260850

Figure Lengend Snippet: PCR results and lineage information associated with the respiratory pathogens used in this study.

Article Snippet: , Influenza A H1N1 , ATCC VR-1683 , Not provided , Not classified , Negative.

Techniques: Virus, Bacteria

Journal: Viruses

Article Title: Efficient Sensing of Avian Influenza Viruses by Porcine Plasmacytoid Dendritic Cells

doi: 10.3390/v3040312

Figure Lengend Snippet: List of influenza A virus (IAV) isolates used in this study.

Article Snippet: HPAIV A/Turkey/Turkey/05, A/Cygnus/Italy/742/06, A/Mallard/Italy/835/06 (H5N1), A/Turkey/Italy/4580/98, A/Ostrich/Italy/2332/00 as well as LPAIV A/Turkey/Italy/3675/99 (H7N1) were kindly provided by Dr. W. Dundon, (IZSV Istituto Zooprofilattico Sperimentale delle Venezie, Venice, Italy), human isolates A/New Caledonia/20/99 (H1N1) A/Wisconsin/67/05 (H3N2) were kindly given by Drs. W. Wunderli and Y. Thomas (National Influenza Reference Center, Geneva University Hospital, Switzerland), swine IAV A/Swine/Belgium/1/98 and A/Swine/Flanders/1/98 (H1N1) were kindly received from Dr. K. Van Reeth (Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Belgium) and A/Hong Kong/8/68 (H3N2) and A/Swine/Iowa/1976/31 (H1N1) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Virus

Journal: Viruses

Article Title: Efficient Sensing of Avian Influenza Viruses by Porcine Plasmacytoid Dendritic Cells

doi: 10.3390/v3040312

Figure Lengend Snippet: Relation between virus dose, pDC percentage and IFN-α response. Porcine pDC were stimulated with H5N1 TT05, H7N1 TI99, H1N1 SI76 and H1N1 NC99 (listed in ) at 100, 1 or 0.01 HAU per well or left non-infected. ( A ) CD4/CD172a plots are shown for H5N1 TT05 infection at the tested doses and for non-infected cells to illustrate the gate for pDC definition. Numbers in the plots indicate the pDC percentage present in the small square. ( B ) pDC percentage after stimulation with different virus doses. ( C ) IFN-α secretion in the supernatant in function of the virus dose.

Article Snippet: HPAIV A/Turkey/Turkey/05, A/Cygnus/Italy/742/06, A/Mallard/Italy/835/06 (H5N1), A/Turkey/Italy/4580/98, A/Ostrich/Italy/2332/00 as well as LPAIV A/Turkey/Italy/3675/99 (H7N1) were kindly provided by Dr. W. Dundon, (IZSV Istituto Zooprofilattico Sperimentale delle Venezie, Venice, Italy), human isolates A/New Caledonia/20/99 (H1N1) A/Wisconsin/67/05 (H3N2) were kindly given by Drs. W. Wunderli and Y. Thomas (National Influenza Reference Center, Geneva University Hospital, Switzerland), swine IAV A/Swine/Belgium/1/98 and A/Swine/Flanders/1/98 (H1N1) were kindly received from Dr. K. Van Reeth (Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Belgium) and A/Hong Kong/8/68 (H3N2) and A/Swine/Iowa/1976/31 (H1N1) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Virus, Infection

Journal: Viruses

Article Title: Efficient Sensing of Avian Influenza Viruses by Porcine Plasmacytoid Dendritic Cells

doi: 10.3390/v3040312

Figure Lengend Snippet: List of reverse genetic viruses used in this study.

Article Snippet: HPAIV A/Turkey/Turkey/05, A/Cygnus/Italy/742/06, A/Mallard/Italy/835/06 (H5N1), A/Turkey/Italy/4580/98, A/Ostrich/Italy/2332/00 as well as LPAIV A/Turkey/Italy/3675/99 (H7N1) were kindly provided by Dr. W. Dundon, (IZSV Istituto Zooprofilattico Sperimentale delle Venezie, Venice, Italy), human isolates A/New Caledonia/20/99 (H1N1) A/Wisconsin/67/05 (H3N2) were kindly given by Drs. W. Wunderli and Y. Thomas (National Influenza Reference Center, Geneva University Hospital, Switzerland), swine IAV A/Swine/Belgium/1/98 and A/Swine/Flanders/1/98 (H1N1) were kindly received from Dr. K. Van Reeth (Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Belgium) and A/Hong Kong/8/68 (H3N2) and A/Swine/Iowa/1976/31 (H1N1) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Binding Assay