c20300 mouse ifn g 96 well white precoated immunospot  (Cellular Technology Ltd)

Journal: iScience

Article Title: Controllable self-replicating RNA vaccine delivered intradermally elicits predominantly cellular immunity

doi: 10.1016/j.isci.2023.106335

Figure Lengend Snippet: c-srRNA vaccine elicits cellular immunity (A–C) Cellular immunity assessed by enzyme-linked immunospot (ELISpot) assays. A single dose or two doses of a placebo (PBO) or 5 μg, or 25 μg of EXG-5003 RNAs, prepared as naked RNAs without LNP or other transfection reagents and in lactated Ringer’s solution, were administered intradermally to BALB/c mice. To assess cellular immunity, an ELISPOT assay, which quantitates the number of cytokine-secreting cells, was performed on splenocytes isolated on Day 14 and Day 42. The splenocytes were stimulated for 24 h with pools of peptides: 9mer peptides for RBD of SARS-CoV-2 (original strain) or 15mer peptides for RBD of SARS-CoV-2 (original strain). Data are presented as mean (SD). (D) Cellular immunity was assessed by intracellular cytokine staining (ICS) and flow cytometry assays. The FACS-ICS assays were performed on splenocytes isolated on Day 42. MFI, mean fluorescence intensity; p, placebo; v, EXG-5003. Data are presented as mean ± SEM. Two-tailed unpaired t-test: ns (p > 0.05), ∗ (p ≤ 0.05), ∗∗ (p ≤ 0.01), ∗∗∗ (p ≤ 0.001), ∗∗∗∗ (p ≤ 0.0001). (E) Cellular immunity assessed by in vivo tumor cell elimination assays. A single EXG-5003 vaccination suppresses the growth of 4T1-LUC tumor cells carrying SARS-CoV-2 spike protein (4T1-LUC-Spike cells). Data are presented as mean (SD).

Article Snippet: Enzyme-linked immunospot (ELISpot) assay , Cellular Technology Limited , Mouse IFN-γ/IL-4 Double-Color ELISPOT 96 well plate white.

Techniques: Enzyme-linked Immunospot, Transfection, Isolation, Staining, Flow Cytometry, Fluorescence, Two Tailed Test, In Vivo

Journal: iScience

Article Title: Controllable self-replicating RNA vaccine delivered intradermally elicits predominantly cellular immunity

doi: 10.1016/j.isci.2023.106335

Figure Lengend Snippet: c-srRNA vaccines can prime the immune system for the subsequent induction of neutralizing antibodies against a variant antigen upon exposure to the variant antigen (A) A schematic diagram of experimental procedures. EXG-5003 RNAs were prepared as naked RNAs, without LNP or other transfection reagents, in lactated Ringer’s solution. (B–D) Groups of CD-1 outbred mice (N = 10) received one of three formulations by intradermal injection on Day 0 and Day 14 (black arrows): (B) a placebo (buffer only), (C) 5 μg of EXG-5003 RNA, or (D) 5 μg of EXG-5003 RNA in combination with an RNase inhibitor (3 units of RNasin Plus; Promega, Madison, WI). Subsequently, all mice received a recombinant RBD protein (Ala319-Phe541, with a C-terminal 6-His tag, NCBI accession number: YP_009724390.1, R&D Systems, Minneapolis, MN) by intradermal injection on Day 49 (open arrows). To assess humoral immunity, an enzyme-linked immunosorbent assay (ELISA), which quantitates the amount of immunoglobulin G (IgG) specific to a recombinant RBD protein (represented as the geometric mean of endpoint titer in triplicate), was performed on serum obtained from the mice on Day −3, Day 14, Day 28, Day 46, Day 56, and Day 63. Data are presented as mean (SD). (E) A schematic diagram of experimental procedures. On day −40, blood was drawn from female BALB/c mice for a plaque reduction neutralization test (PRNT). On day −36, these mice received an intradermal injection of EXG-5003 RNA, which was prepared as a naked RNA, without an LNP or transfection reagent. On day −22 (14 days after EXG-5003 vaccination), half of the mice were sacrificed to obtain splenocytes for ELISpot assays. On day 0, the remaining mice were intradermally injected with the spike protein of the SARS-CoV-2 delta variant (B.1.617.2: R&D Systems) mixed with adjuvant—AddaVax (Invivogen). On day 7 (7 days after the spike protein injection), blood was drawn for the PRNT assay. (F) The induction of cellular immunity against the RBD protein by a single intradermal administration of EXG-5003 RNA. The ELISpot assay shows the number of IFN-γ spot-forming cells (SFCs) in 1 × 10ˆ6 splenocytes from immunized mice restimulated by culturing in the presence or absence of a pool of 15mer peptides for RBD of SARS-CoV-2 (original strain). The number of SFC obtained in the presence of peptides was plotted on the graph after subtracting the number of SFC obtained in the absence of peptides (background). The average and standard deviation (error bars) of five mice (n = 5) are shown for each group. (G) The titer of serum antibodies that can neutralize (50%) the SARS-CoV-2 virus (delta variant B.1.617.2), measured by the PRNT assay. Exposure to the spike protein of the SARS-CoV-2 virus (delta variant B.1.617.2) induced neutralization antibodies against the delta variant of the SARS-CoV-2 virus only in mice vaccinated with EXG-5003, which encodes the RBD of SARS-CoV-2 (original strain). Data are presented as mean (SD).

Article Snippet: Enzyme-linked immunospot (ELISpot) assay , Cellular Technology Limited , Mouse IFN-γ/IL-4 Double-Color ELISPOT 96 well plate white.

Techniques: Vaccines, Variant Assay, Transfection, Injection, Recombinant, Enzyme-linked Immunosorbent Assay, Plaque Reduction Neutralization Test, Enzyme-linked Immunospot, Adjuvant, Standard Deviation, Virus, Neutralization

Journal: iScience

Article Title: Controllable self-replicating RNA vaccine delivered intradermally elicits predominantly cellular immunity

doi: 10.1016/j.isci.2023.106335

Figure Lengend Snippet: Improvement of c-srRNA and its use as a booster vaccine to induce antibodies against an antigen, following earlier administration of the antigen protein (A) Comparison of srRNA and c-srRNAs for T cell-inducibility. A schematic diagram of experimental procedures. On day 0, mice were intradermally injected with either placebo (PBO, buffer only), srRNA0, c-srRNA1, or c-srRNA3. The srRNA0, c-srRNA1, and c-srRNA3 encode the same RBD of SARS-CoV-2 (original strain). On day 14, mice were sacrificed and splenocytes were isolated for ELISpot assays. (B) The ELISpot assay shows the number of IFN-γ spot-forming cells (SFCs) in 1 × 10ˆ6 splenocytes from immunized mice restimulated by culturing in the presence or absence of a pool of 15mer peptides for RBD of SARS-CoV-2 (original strain). The number of SFC obtained in the presence of peptides was plotted on the graph after subtracting the number of SFC obtained in the absence of peptides (background). The average and standard deviation (error bars) are shown for each group. Two-tailed unpaired t test: ns (p > 0.05), ∗ (p ≤ 0.05), ∗∗ (p ≤ 0.01), ∗∗∗∗ (p ≤ 0.0001). (C) Use of c-srRNA vaccine as a booster to induce antibodies against an antigen, following earlier administration of the antigen protein. A schematic diagram of experimental procedures. On day 0 (first treatment), female C57BL/6 mice were treated with an intradermal injection of 10 μg RBD protein (Sino Biological SARS-CoV-2 [original strain]) + adjuvant (AddaVax). On day 14 (second treatment), the mice were treated with an intradermal injection of a placebo (PBO: buffer only), 25 μg EXG-5003 (c-srRNA1 encoding RBD [original strain]), 25 μg of EXG-5003o (c-srRNA3 encoding RBD [omicron variant B.1.1.529 BA.1]), or 10 μg RBD protein (Sino Biological SARS-CoV-2) + adjuvant (AddaVax). On day 28, the mice were sacrificed, and splenocytes and serum were collected for ELISpot and ELISA assays. (D) The induction of cellular immunity is shown by the frequency of IFN-γ spot-forming cells (SFC) in 1 × 10ˆ6 splenocytes restimulated by culturing in the presence or absence of a pool of 15mer peptides for RBD of SARS-CoV-2 (original strain) (RBD-PBO, RBD-EXG-5003, RBD-RBD) or 15mer peptides for RBD of SARS-CoV-2 (omicron variant) (RBD-EXG-5003o). The frequency obtained in the presence of peptides is plotted in the graph after subtracting the frequency obtained in the absence of peptides (background). Data are represented as mean (SD). (E) The levels of serum antibodies against the RBD protein of the SARS-CoV-2 (original strain), measured by an ELISA assay. The levels of antibodies are represented by the OD450 measurement. The average and standard deviation (error bars) of five mice (n = 5) are shown for each group. The data from Day −1 (before the first treatment) and the data from Day 28 (after the second treatment) are shown for each group. Data are represented as mean (SD).

Article Snippet: Enzyme-linked immunospot (ELISpot) assay , Cellular Technology Limited , Mouse IFN-γ/IL-4 Double-Color ELISPOT 96 well plate white.

Techniques: Comparison, Injection, Isolation, Enzyme-linked Immunospot, Standard Deviation, Two Tailed Test, Adjuvant, Variant Assay, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Controllable self-replicating RNA vaccine delivered intradermally elicits predominantly cellular immunity

doi: 10.1016/j.isci.2023.106335

Figure Lengend Snippet: Use of nucleoprotein as an antigen and test for the requirement of signal peptides (A) A schematic drawing of antigen structure. EXG-5004: c-srRNA3 encodes the nucleoprotein (N) protein of SARS-CoV-2 (original strain). EXG-5005: c-srRNA3 encodes a fusion protein of signal peptide sequence of the human CD5 protein and the nucleoprotein (N) protein of SARS-CoV-2 (original strain). (B–G) Cellular immunity by ELISpot assays, showing the frequency of IFN-γ spot-forming cells (SFC) or IL-4 SFC in 1 × 10ˆ6 splenocytes obtained from CD-1 outbred mice or BALB/c inbred mice that were immunized by a single intradermal injection of 100 μL solution containing either 5 μg or 25 μg of EXG-5004, EXG-5005, or a placebo (PBO: buffer only). The splenocytes were restimulated by culturing in the presence and absence of a pool of 15mer peptides for nucleoprotein of SARS-CoV-2 (original strain). The frequency obtained in the presence of peptides is plotted in the graph after subtracting the frequency obtained in the absence of peptides (background). The average and standard deviation (error bars) of five mice (n = 5) are shown for each group. (B) CD-1 mouse, EXG-5004, IFN-γ. (C) CD-1 mouse, EXG-5004, IL-4. (D) CD-1 mouse, EXG-5005, IFN-γ. (E) CD-1 mouse, EXG-5005, IL-4. (F) BALB/c mouse, EXG-5005, IFN-γ. (G) BALB/c mouse, EXG-5005, IL-4.

Article Snippet: Enzyme-linked immunospot (ELISpot) assay , Cellular Technology Limited , Mouse IFN-γ/IL-4 Double-Color ELISPOT 96 well plate white.

Techniques: Sequencing, Enzyme-linked Immunospot, Injection, Standard Deviation

Journal: iScience

Article Title: Controllable self-replicating RNA vaccine delivered intradermally elicits predominantly cellular immunity

doi: 10.1016/j.isci.2023.106335

Figure Lengend Snippet: EXG-5006, encoding a fusion protein of SARS-CoV-2 and MERS-CoV nucleoproteins, can eliminate cells expressing those antigens in mouse (A) A schematic drawing of EXG-5006, encoding a fusion protein of CD5 signal peptide, nucleoprotein of SARS-CoV-2, and a nucleoprotein of MERS-CoV. (B–E) Cellular immunity by ELISpot assays, showing the frequency of IFN-γ (B, D) spot-forming cells or IL-4 (C and E) spot-forming cells in 1 × 10ˆ6 splenocytes obtained from BALB/c mice on Day 14 after vaccinating by a single intradermal injection of 100 μL solution containing either 5 μg (n = 1) or 25 μg (n = 4) of EXG-5006, or a placebo (PBO: buffer only: n = 5). The splenocytes were restimulated by culturing in the presence and absence of a pool of 15mer peptides for nucleoprotein of SARS-CoV-2 (original strain) (B, C) or a pool of 15mer peptides for nucleoprotein of MERS-CoV (D, E). The frequency obtained in the presence of peptides is plotted in the graph after subtracting the frequency obtained in the absence of peptides (background). Data are presented as mean (SD). (F) shows the survival (%) of female BALB/c mice vaccinated with EXG-5006, followed by injection of 4T1 tumor cells expressing the same antigen (A fusion protein of nucleoproteins of SARS-CoV-2 and MERS-CoV, without CD5 signal peptides).

Article Snippet: Enzyme-linked immunospot (ELISpot) assay , Cellular Technology Limited , Mouse IFN-γ/IL-4 Double-Color ELISPOT 96 well plate white.

Techniques: Expressing, Enzyme-linked Immunospot, Injection

Journal: iScience

Article Title: Controllable self-replicating RNA vaccine delivered intradermally elicits predominantly cellular immunity

doi: 10.1016/j.isci.2023.106335

Figure Lengend Snippet: Generation of EXG-5008, encoding RBD and nucleoproteins of SARS-CoV-2 and MERS (A) A schematic diagram of EXG-5008, encoding a fusion protein of the signal peptide of CD5, the RBD and nucleoprotein of SARS-CoV-2, and the RBD and nucleoprotein of MERS-CoV. (B and C) The frequency of IFN-γ spot-forming cells (B) and the frequency of IL-4 spot-forming cells (C) in 1 × 10ˆ6 splenocytes obtained from female C57BL/6 mice that were immunized by a single intradermal injection of 100 μL solution containing either placebo (PBO: buffer only), 25 μg of EXG-5006a, or 25 μg of EXG-5008. The splenocytes were restimulated by culturing in the presence or absence of pools of peptides: 15mer peptides for RBD of SARS-CoV-2 (original strain) ( 1 ); 15mer peptides for nucleoprotein of SARS-CoV-2 (original strain) ( 2 ); 15mer peptides for nucleoprotein of MERS-CoV ( 3 ); 15mer peptides for the spike of MERS-CoV ( 4 ). The cellular immunity was analyzed by ELISpot assays. The frequency obtained in the presence of peptides is plotted in the graph after subtracting the frequency obtained in the absence of peptides (background). The average and standard deviation (error bars) of five mice (n = 5) for PBO, four mice (n = 4) for EXG-5006, and five mice (n = 5) for EXG-5008, are shown for each group. Splenocytes were isolated 14 days after vaccination. (D) A model showing how the c-srRNA booster vaccine works. Primary series of vaccines or prior infections expose naive B cells to viral surface proteins and turn them into memory B cells in a manner dependent on CD4 + helper T cells. Skin delivery of a c-srRNA booster vaccine encoding a fusion antigen of the viral surface proteins and internal proteins, whose sequences are more evolutionarily conserved than the surface proteins, is primarily incorporated into skin antigen-presenting cells such as Langerhans cells and dendritic cells. Within the antigen-presenting cells, c-srRNA replicates and produces the fusion antigen. The antigen-presenting cells digest the antigen into peptides and present these peptides to T cells. The peptides presented through this pathway stimulate MHC-I-restricted CD8 + cytotoxic T cells as well as MHC-II-restricted CD4 + helper T cells. CD8 + cytotoxic T cells eliminate infected cells (B) CD4 + helper T cells stimulate the memory B cells and enhance or restore the production of neutralizing antibody, which prevents infection.

Article Snippet: Enzyme-linked immunospot (ELISpot) assay , Cellular Technology Limited , Mouse IFN-γ/IL-4 Double-Color ELISPOT 96 well plate white.

Techniques: Injection, Enzyme-linked Immunospot, Standard Deviation, Isolation, Vaccines, Infection

Journal: iScience

Article Title: Controllable self-replicating RNA vaccine delivered intradermally elicits predominantly cellular immunity

doi: 10.1016/j.isci.2023.106335

Figure Lengend Snippet:

Article Snippet: Enzyme-linked immunospot (ELISpot) assay , Cellular Technology Limited , Mouse IFN-γ/IL-4 Double-Color ELISPOT 96 well plate white.

Techniques: Virus, Recombinant, Modification, Derivative Assay, Variant Assay, Saline, Plasmid Preparation, Enzyme-linked Immunospot, Double-Color Enzymatic ELISPOT, Enzyme-linked Immunosorbent Assay, Plaque Reduction Neutralization Test, Software, Imaging

Journal: Journal of Biomedicine and Biotechnology

Article Title: Immunogenicity of a Recombinant Influenza Virus Bearing Both the CD4+ and CD8+ T Cell Epitopes of Ovalbumin

doi: 10.1155/2011/497364

Figure Lengend Snippet: T cell responses in WSN-OVA I/II virus-infected mice. C57BL/6J mice were immunized i.n. with 5 × 10 2 PFU of WSN virus or 3 × 10 5 PFU of WSN-OVA I/II virus, and sacrificed on day 9 for measuring epitope-specific CD8+ T cell responses in mediastinal lymph nodes (MLN) and spleens (a) and CD4+ T cell responses in MLN (b), respectively, by use of an IFN- γ ELISPOT assay with the indicated peptides. The values presented are averages of triplicate wells; the error bars indicate the standard deviation. The data are representative of three experiments. * P < 0.05 comparing WSN- versus WSN-OVA I/II -infected mice.

Article Snippet: Nine days later, five mice from each group were sacrificed to obtain spleens and mediastinal lymph nodes (MLN), and epitope-specific T cell responses were determined by using a mouse IFN- γ enzyme-linked immunospot assay (ELISPOT) kit (Pharmingen, BD).

Techniques: Infection, Enzyme-linked Immunospot, Standard Deviation

Journal: Journal of Biomedicine and Biotechnology

Article Title: Immunogenicity of a Recombinant Influenza Virus Bearing Both the CD4+ and CD8+ T Cell Epitopes of Ovalbumin

doi: 10.1155/2011/497364

Figure Lengend Snippet: Protective and antigen-specific immunity to melanoma B16 cells expressing OVA. C57BL/6J mice were immunized once with either the parental WSN virus or the recombinant WSN-OVA I/II virus. Three weeks after immunization, mice were challenged i.v. with 3 × 10 5 B16-OVA cells or with PBS. The number of pulmonary metastases (a) and the frequency of SIINFEKL-specific IFN- γ -producing T cells, as measured by using the ELISPOT assay on spleen-derived lymphocytes (b) was determined 12 days after the tumor cell inoculation. All of the experiments included 8 mice per group and were repeated three times with similar results. * P < 0.001 and ** P < 0.0001, comparing WSN-OVA I/II -versus WSN-infected and naïve mice.

Article Snippet: Nine days later, five mice from each group were sacrificed to obtain spleens and mediastinal lymph nodes (MLN), and epitope-specific T cell responses were determined by using a mouse IFN- γ enzyme-linked immunospot assay (ELISPOT) kit (Pharmingen, BD).

Techniques: Expressing, Recombinant, Enzyme-linked Immunospot, Derivative Assay, Infection

Journal: PLoS ONE

Article Title: A Whole Virus Pandemic Influenza H1N1 Vaccine Is Highly Immunogenic and Protective in Active Immunization and Passive Protection Mouse Models

doi: 10.1371/journal.pone.0009349

Figure Lengend Snippet: Balb/c mice were immunized with pandemic H1N1 (H1N1 A/California/7/2009), seasonal H1N1 (H1N1 A/Brisbane/59/2007), and pandemic H5N1 (H5N1 A/Vietnam/1203/2004) vaccines. Spleen cells were collected 7 days after the first, or 21 days after the booster immunization (i.e. 42 days after the first), and stimulated with various seasonal or pandemic influenza virus antigens, before determination of cells responding by secretion of either IFN-g or IL-4 by an ELISPOT assay. Anti-HA IgG subclass responses were analyzed by ELISA using sera collected on day 42.

Article Snippet: The frequency of IFN-γ- or Interleukin-4 (IL-4)-secreting cells was analyzed using mouse IFN-γ and IL-4 enzyme-linked immunospot (ELISPOT) kits (Mabtech AB, Nacka, Sweden), as previously described , using vaccine antigen at a concentration of 0.3 µg HA/ml.

Techniques: Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay