Journal: mBio

Article Title: Surface-modified measles vaccines encoding oligomeric, prefusion-stabilized SARS-CoV-2 spike glycoproteins boost neutralizing antibody responses to Omicron and historical variants, independent of measles seropositivity

doi: 10.1128/mbio.02928-23

Figure Lengend Snippet: Poor pseudovirus-neutralizing antibody production and absence of T-cell responses to full-length SARS-CoV-2 spike ectodomain protein vaccination in IFNAR −/− -CD46Ge mice. ( A ) IFNAR −/− -CD46Ge mice were vaccinated intraperitoneally twice with 5 µg of alum-adjuvanted full-length spike ectodomain (S1 + S2), spike receptor-binding domain (S1-RBD), spike S1 domain (S1), spike S2 domain (S2), and nucleocapsid (N). Serum samples were collected on days 21 and 49, and the levels of spike ectodomain-binding IgG antibodies were quantified by enzyme-linked immunosorbent assay. ( B ) The binding IgG in serum was also quantified by ELISA for binding to homologous or heterologous antigens ( x -axis). ( C ) Pseudovirus-neutralizing antibody titers were determined using pseudotyped viruses expressing the SARS-CoV-2 spike protein bearing the D614G amino acid change. ( D ) T-cell responses elicited against SARS-CoV-2 spike were determined by an enzyme-linked immunospot assay. The data are presented as IFN-γ-secreting cells or spot-forming cells per 1 × 10 6 splenocytes, with values representing the geometric mean ± geometric standard deviation. Statistical significance was determined using two-way ANOVA with Bonferroni’s multiple comparison test (*, P < 0.05; **, P < 0.005; ***, P < 0.0004; ****, P < 0.0001), and only significant values are shown.

Article Snippet: The developed IFN-γ spots were counted with an automated ELISpot reader (CTL Analyzers LLC, USA).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Enzyme-linked Immunospot, Standard Deviation, Comparison

Journal: mBio

Article Title: Surface-modified measles vaccines encoding oligomeric, prefusion-stabilized SARS-CoV-2 spike glycoproteins boost neutralizing antibody responses to Omicron and historical variants, independent of measles seropositivity

doi: 10.1128/mbio.02928-23

Figure Lengend Snippet: Trimerization and stabilization of SARS-CoV-2 spike protein constructs enhance humoral antibody and T-cell responses. ( A ) Schematic of the MeV-MR vector and various SARS-CoV-2 spike-based constructs inserted as an additional transcript unit, showing modifications to the transmembrane (TM) and cytoplasmic tail regions and substitution of the spike signal peptide with a murine IgG kappa leader sequence plus an HA tag. The H. pylori NAP was genetically fused at the C-terminus of the spike protein, optionally incorporating a stop termination codon or a foldON trimerization domain. ( B and C ) IgG-binding responses in IFNAR −/− -CD46Ge mice vaccinated with different recombinant viruses, as measured by ELISA for binding to ( B ) MeV-bulk antigen and ( C ) the spike ectodomain. ( D ) Pseudovirus-neutralizing antibody responses in mice vaccinated once or twice with the various recombinant viruses, as determined by pseudotyped lentiviruses expressing the SARS-CoV-2 spike D614G construct. ( E ) IFN-γ enzyme-linked immunospot assay for T-cell responses, showing the number of spot-forming cells per 1 × 106 splenocytes isolated from mice vaccinated twice and stimulated ex vivo with phorbol myristate acetate/ionomycin or antigen-specific peptides. Values are represented as the geometric mean ± geometric standard deviation, with each data point representing an individual mouse. Statistical significance was determined using one-way or two-way ANOVA with Bonferroni’s multiple comparison test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Article Snippet: The developed IFN-γ spots were counted with an automated ELISpot reader (CTL Analyzers LLC, USA).

Techniques: Construct, Plasmid Preparation, Sequencing, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Expressing, Enzyme-linked Immunospot, Isolation, Ex Vivo, Standard Deviation, Comparison

Journal: Immunology

Article Title: Artificial human antigen‐presenting cells are superior to dendritic cells at inducing cytotoxic T‐cell responses

doi: 10.1111/imm.12783

Figure Lengend Snippet: Artificial antigen‐presenting cells (aAPCs) (NP‐Mart‐1) are superior to mature dendritic cells (mDCs) ‐Mart‐1 when presenting antigen and inducing cytotoxic T‐lymphocyte (CTL) response. ELISpot and cytotoxicity assays were performed to assess the Mart‐1 antigen presentation and CTL cytotoxic activities. Both the ELISpot and cytotoxicity assays were independently repeated three times, and each condition was performed in triplicate. (a) Representative ELISpot images. (b) Comparison of the Mart‐1 antigen presentation between mDC‐Mart‐1 and aAPCs (NP‐Mart‐1) using the ELISpot assay. Empty aAPCs (mDCs + CNPs) and mDCs were also included as the controls. Significantly more Mart‐1 antigen was presented to the TIL 1235 cells by the aAPCs (NP‐Mart‐1) than by the mDC‐Mart‐1 (P = 0·0004). (c) aAPCs presented antigen to T cells in a sustained manner. ELISpot assays were performed at different incubation times of 24, 36 and 48 hr. Interferon‐γ (IFN‐γ) spots in the aAPC group were more significant than those in the peptide‐pulsed mDCs group at the above incubation time‐points (P < 0·001). (d) The Mart‐1‐specific CTLs generated by both aAPCs (NP‐Mart‐1) and mDC‐Mart‐1 were capable of lysing Mart‐1 peptide‐pulsed T2 cells [solid green line and solid red line in (c)]. The cytotoxic activity of the CTLs generated with the aAPCs (NP‐Mart‐1) was significantly higher than that of the CTLs generated with mDC‐Mart‐1 (P values of 0·007, 0·001 and 0·012 for the E : T ratios of 50 : 1, 25 : 1 and 12·5 : 1, respectively). (e) The Mart‐1‐specific CTLs generated by both aAPCs (NP‐Mart‐1) and mDC‐Mart‐1 were also able to lyse melanoma 624 cells [solid green line and solid pink line in (e)]. The cytotoxic activity of the CTLs generated with aAPCs (NP‐Mart‐1) was significantly higher than that of the CTLs generated by mDC‐Mart‐1 (P values of 0·009, 0·001 and 0·005 for the E : T ratios of 50 : 1, 25 : 1 and 12·5 : 1, respectively). In addition, the CTLs induced by aAPCs (CNPs) were also included as a control, and the results showed that they failed to recognize and kill the target cells of both T2 cells and Mart‐1 peptide‐pulsed T2 cells [solid and broken blue lines in (d)]. Each assay was independently repeated three times, and each condition was performed in triplicate. [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Finally, the IFN‐ γ ‐secreting T cells (spot‐forming cells) on the membrane were counted using an ELISpot reader system (CTL Analyzers LLC, Cleveland, OH).

Techniques: Enzyme-linked Immunospot, Incubation, Generated, Activity Assay

Journal: STAR Protocols

Article Title: FluoroSpot assay to analyze SARS-CoV-2-specific T cell responses

doi: 10.1016/j.xpro.2023.102584

Figure Lengend Snippet:

Article Snippet: S6 Ultra M2 analyzer , CTL Analyzers, LLC , N/A.

Techniques: Recombinant, Synthesized, Saline, Staining, Software