soluble gp1–gp2 fusion protein (linked lasv-gp, lineage iv)  (Zalgen Labs)

Journal: Communications Medicine

Article Title: The DNA-based Lassa vaccine INO-4500 confers durable protective efficacy in cynomolgus macaques against lethal Lassa fever

doi: 10.1038/s43856-024-00684-8

Figure Lengend Snippet: a NHPs were divided into four groups of 6 animals/group. Animals received 2, 1 or 0.1 mg of INO-4500 (with intradermal (ID) EP; groups 1, 2 and 3, respectively), and a fourth group served as unvaccinated control. NHPs were dosed at weeks 0 and 4, after which animals were challenged with LASV Josiah at week 8. Serum and PBMCs were collected to assess the presence of LASV-specific antibody and T cell responses before and after immunizations. b LASV-specific antibody responses were assessed at week 6 by IgG Binding ELISA to lineage IV Prefusion GPC. IgG titers were calculated based on the highest serum dilution with OD 450 equal to or higher than 0.150 times 2.5 standard deviations of the experimental duplicates. Error bars represent the geometric mean (GMT) ± 95% confidence interval (CI). c IFNγ T cell responses at week 6 for each group was measured in PBMCs stimulated with lineage IV Josiah GPC peptide megapools by IFNγ ELISpot assay. Data were calculated after DMSO subtraction from peptide-stimulated wells. Error bars represent the mean ± standard error of the mean (SEM). b-c Each datapoint represents the mean of technical duplicates for each NHP. d Kaplan-Meier survival curves for vaccinated and control animals after challenge with 1000 PFU LASV Josiah at week 8. e Quantification of LASV Josiah in PFU per mL of serum by group. Serum samples were collected every 3–5 days to monitor viremia in vaccinated and control NHPs. Surviving NHPs were monitored until Day 38 post-challenge. Shaded areas indicate viral levels above 4 log10 PFU/mL, considered to be peak at critical phase. Animals noted with a black X indicate vaccinated NHPs that were euthanized during critical phase. f LASV neutralization in serum as measured by plaque reduction neutralization test (PRNT).

Article Snippet: To determine antigen-specific antibody responses in sera, 96-well ELISA plates were coated with 1 µg/ml recombinant LASV lineage IV GP prefusion protein (Zalgen Labs) or 1 µg/ml recombinant LASV lineages I, II and III GP prefusion protein (kindly donated from Dr. Erica Sapphire, La Jolla Institute for Immunology) in 1x PBS overnight at 4 °C.

Techniques: Control, Binding Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Neutralization, Plaque Reduction Neutralization Test

Journal: Communications Medicine

Article Title: The DNA-based Lassa vaccine INO-4500 confers durable protective efficacy in cynomolgus macaques against lethal Lassa fever

doi: 10.1038/s43856-024-00684-8

Figure Lengend Snippet: a NHPs were divided into four groups of 6 animals/group. Animals received one or two 2 mg doses of INO-4500 with ID-EP at week 0 or weeks 0 and 4, respectively. The third group served as control. All animals were challenged at week 8 with a target of 1000 PFU LASV Josiah. Serum and PBMCs were collected to assess the presence of LASV-specific antibody and T cell responses before and after immunizations. b LASV-specific antibody response was assessed by IgG Binding ELISA to LASV lineage IV Prefusion GPC at weeks 4 and 6. IgG titers were calculated based on the highest serum dilution with OD 450 equal to or higher than 0.250 + 2.5 standard deviations of the experimental duplicates. Error bars represent the GMT ± 95% CI. c IFNγ responses at week 4 and 6 for each group were measured in PBMCs stimulated with lineage IV Josiah GPC peptide megapools by IFNγ ELISpot assay. Data were calculated after DMSO subtraction from peptide-stimulated wells. Error bars represent the mean ± SEM. b , c Each datapoint represents the mean of technical duplicates for each NHP. d Kaplan-Meier survival curves for vaccinated and control animals after challenge with 1000 PFU LASV Josiah at week 8. e Quantification of LASV in PFU per mL of serum by group. Serum samples were collected every 3–5 days to monitor viremia in vaccinated and control NHPs. In NHPs that succumbed to disease, viremia was monitored until the time of death or euthanasia. Surviving NHPs were monitored until Day 30 post-challenge. Shaded areas indicate peak viremia levels above 4 log 10 PFU/mL at critical phase. Animals noted with a black X indicate vaccinated NHPs that succumbed to disease. f LASV neutralization in serum as measured by plaque reduction neutralization test (PRNT).

Article Snippet: To determine antigen-specific antibody responses in sera, 96-well ELISA plates were coated with 1 µg/ml recombinant LASV lineage IV GP prefusion protein (Zalgen Labs) or 1 µg/ml recombinant LASV lineages I, II and III GP prefusion protein (kindly donated from Dr. Erica Sapphire, La Jolla Institute for Immunology) in 1x PBS overnight at 4 °C.

Techniques: Control, Binding Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Neutralization, Plaque Reduction Neutralization Test

Journal: Communications Medicine

Article Title: The DNA-based Lassa vaccine INO-4500 confers durable protective efficacy in cynomolgus macaques against lethal Lassa fever

doi: 10.1038/s43856-024-00684-8

Figure Lengend Snippet: a Overview of study Arm 3. NHPs were immunized with INO-4500 + ID-EP at weeks 0 and 4. NHPs were challenged with LASV at week 59. b LASV-specific IgG responses by IgG Binding ELISA to LASV Clade IV Prefusion GPC for 2 mg and 1 mg groups. LASV IgG antibody response was assessed at baseline, after two immunizations at week 6, and prior to LASV challenge at week 54. Error bars represent the GMT ± 95% CI. c Individual IFNγ cellular responses after INO-4500 immunization for NHPs from 2 mg and 1 mg groups. PBMCs were isolated at baseline, at week 6 and prior to LASV challenge at week 54. PBMCs were stimulated with Clade IV Josiah GPC peptide megapools. Data were calculated after DMSO subtraction from peptide-stimulated wells. Error bars represent the mean ± SEM. b-c Each datapoint represents the mean of technical duplicates for each NHP. Quantification of d LASV-specific CD4+ and e CD8 + T cells upon stimulation with peptide megapools spanning the Josiah lineage IV GPC. Cells were stained with antibodies against markers that are upregulated upon T cell activation, including CD69, CD134, CD137, CD154, and CD25. Frequency of LASV + T cells were calculated based on any T cell expressing at least two AIM markers. Percentage of positive CD4+ and CD8 + T cells were calculated after DMSO subtraction from peptide-stimulated samples. Bars represent the mean ± SD. Each symbol represents an individual NHP test sample. *p ≤ 0.05, Two-way ANOVA multiple comparison. f Kaplan-Meier survival curves following LASV challenge with Josiah strain one year post-initial immunization.

Article Snippet: To determine antigen-specific antibody responses in sera, 96-well ELISA plates were coated with 1 µg/ml recombinant LASV lineage IV GP prefusion protein (Zalgen Labs) or 1 µg/ml recombinant LASV lineages I, II and III GP prefusion protein (kindly donated from Dr. Erica Sapphire, La Jolla Institute for Immunology) in 1x PBS overnight at 4 °C.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Isolation, Staining, Activation Assay, Expressing, Comparison