Journal: American Journal of Cancer Research

Article Title: Immune response evoked by tumor-associated NADH oxidase (tNOX) confers potential inhibitory effect on lung carcinoma in a mouse model

doi:

Figure Lengend Snippet: The protein expression and immune responses of the human tNOX recombinant proteins in mice. Recombinant human tNOX was expressed in E. coli and purified with His-tag purification resin. A. The expressed, purified and dialyzed proteins were resolved by SDS-PAGE and detected by western blot analysis with anti-His tag and anti-tNOX antibodies, and are shown in Lanes 1, 2 and 3, respectively. The molecular weight (KDa) marker (M) is shown at the left. Arrow indicates the tNOX protein band. B. Blood samples were collected at 0, 14, 28 and 35 days after the first vaccination and antibody titers were measured by ELISA. The specific anti-tNOX antibody response was significantly higher in the tNOX vaccine group compared with unvaccinated controls (*P < 0.05). C. Splenocytes of mice were stimulated with human tNOX protein and IFN-γ-secreting T cells were evaluated by ELISpot assay. Representative images show proliferative spots of splenocytes from mice vaccinated with tNOX (tNOX) or without tNOX (NC). D. The frequencies of tNOX-specific IFN-γ-secreting T cells was significantly higher in the tNOX vaccine group compared to the NC group (*P < 0.05). The average spot counts of the NC and tNOX vaccine groups were 67 ± 13 and 155 ± 42, respectively.

Article Snippet: ELISpot assays were performed using a Mouse IFN-γ ELISpot Development Module (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s protocol.

Techniques: Expressing, Recombinant, Purification, SDS Page, Western Blot, Molecular Weight, Marker, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: FASEB bioAdvances

Article Title: AJP001, a novel helper T‐cell epitope, induces a humoral immune response with activation of innate immunity when included in a peptide vaccine

doi: 10.1096/fba.2019-00056

Figure Lengend Snippet: Vaccination with AJP001 induces antibody production and T‐cell activation in mice. A. Ang II, a mixture of Ang II and AJP001 or the AJP001‐Ang II conjugate vaccine was administered intracutaneously to 7‐week‐old female BALB/cA three times at 2‐week intervals. The anti‐Ang II IgG antibody titer in serum samples collected at 0, 2, 4, 6, and 8 weeks was detected by ELISA. The data represent the mean OD at 450 nm and the SD at each serum dilution fold (n = 3). B, C. The AJP001‐Ang II conjugate vaccine (AJP001‐Ang II 100 μg) was administered intracutaneously to 7‐week‐old female BALB/cA (B) or 7‐week‐old male C57BL/6 (C) mice three times at 2‐week intervals without any adjuvant cotreatment. The anti‐Ang II IgG antibody titer in serum sample collected at 0, 2, 4, 6, and 8 weeks was detected by ELISA. The data represent the mean OD at 450 nm and the SD at each serum dilution fold (n = 3). D. The AJP001‐Ang II conjugate vaccine was administered intracutaneously to 7‐week‐old female BALB/cA mice at a dose of 20, 100, or 500 μg per mouse three times at 2‐week intervals without any adjuvant cotreatment. The anti‐Ang II IgG antibody titer in serum samples collected at 0, 2, 4, 6, and 8 weeks was measured by ELISA. The titers are expressed as the dilution fold of the serum giving half‐maximal absorbance at 450 nm. All data are expressed as the mean ± SD (n = 3). * P < .05, ** P < .01 and *** P < .001 vs the saline group. E, F. Antigen‐specific activation of T cells in AJP001‐Ang II‐immunized mice was evaluated by an ELISpot assay. Splenocytes were isolated from AJP001‐Ang II‐immunized mice and stimulated with Angiotensin II or AJP001 at a concentration of 10 μg/mL. PMA and ionomycin (100 ng/m each) were added to positive control wells, and medium was added as a negative control. The number of IFN‐γ‐ (E) or IL‐4‐producing (F) cells was detected by counting spots using a stereomicroscope. The number of spots was quantified in the duplicate or triplicate wells of each mouse. The data represent the mean ± SD (n = 3). * P < .05 and ** P < .01 vs the saline group

Article Snippet: Mouse IFN‐γ ELISpot Development Module, Mouse IL‐4 ELISpot Development Module and ELISpot Blue Color Module were obtained from R&D Systems, Inc.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Adjuvant, Saline, Enzyme-linked Immunospot, Isolation, Concentration Assay, Positive Control, Negative Control

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Effective Depletion of Pre-existing Anti-AAV Antibodies Requires Broad Immune Targeting

doi: 10.1016/j.omtm.2017.01.003

Figure Lengend Snippet: Differential Impacts of Rap + Pred on Bone Marrow Antibody-Secreting Cells (A–E) WT mice were immunized with an i.p. injection of rAAV9 vector, and then they were treated with rapamycin (R, 2 mg/kg, every other day) and prednisolone (P, 0.75 mg/kg, daily) via i.p. injection (R + P), beginning at 4 weeks post-immunization. Controls were matched naive and AAV9-immunized mice without IS treatment. Bone marrow (BM) cells were assayed at 8 weeks of IS treatment by ELISpot for (A, B, and D) IgG-secreting and (C and E) AAV9-Ab-secreting plasma cells (PCs). (B and C) p = 0.04 and p = 0.13 R + P versus non-IS. Naive, non-immunized WT mice; non-IS, non-IS-treated AAV9-immunized mice; R + P, AAV9-immunized mice treated with R + P.

Article Snippet: To assess a frequency of antibody-secreting cells (ASCs) among total bone marrow cells, IgG-ELISpot assays were performed using Mouse IgG B cell ELISpot kits (SELB004 and SEL002, R&D Systems), following the procedures provided by the manufacturer.

Techniques: Injection, Plasmid Preparation, Enzyme-linked Immunospot, Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: Exploring in vitro expression and immune potency in mice using mRNA encoding the Plasmodium falciparum malaria antigen, CelTOS

doi: 10.3389/fimmu.2022.1026052

Figure Lengend Snippet: Effect of N-glycosylation on in vitro and in vivo responses. (A) Western blot analysis of CHO cells transfected with mRNAs pfceltos ARCA, pfceltos Cap1 and pfceltos ARCA GM. pfceltos mRNA transcripts were codon harmonized (CH) for optimal expression in mice and encoded with the native falciparum celtos signal sequence (Wt-SS). Cell culture supernatants and cell lysates were harvested at 24 hours post-transfection to assess for the effect of N-glycosylation on protein translation. (B) BALB/cJ mice were immunized intramuscularly (IM) two times at a three-week interval with 10µg of pfceltos mRNA encapsulated into LNP1 (10µg LNP1), 10µg pfceltos mRNA with glycosylation site modified (GM) in LNP1 (10µg GM LNP1) or LNP1 alone ( n= 5 per group). PfCelTOS-specific IgG antibody concentrations (µg/mL) were quantified in sera at pre-immunization, three weeks after the primary dose and two weeks after the final dose by ELISA. Antibody concentrations are reported as the geometric mean and 95% confidence intervals. (C, D) Splenocytes were harvested and IFN-γ and IL-4 cytokines were detected by ELISpot assay. The mean number of spot-forming cells (SFC) per splenocytes were reported with standard errors of the mean (SEM). Statistical analysis was performed using Mann-Whitney test (**p<0.01).

Article Snippet: To determine cellular responses against PfCelTOS, mouse IFN-γ and IL-4 ELISpot assay (R&D systems SEL485 SEL404, respectively) were performed according to the manufacturer’s instructions.

Techniques: In Vitro, In Vivo, Western Blot, Transfection, Expressing, Sequencing, Cell Culture, Modification, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Exploring in vitro expression and immune potency in mice using mRNA encoding the Plasmodium falciparum malaria antigen, CelTOS

doi: 10.3389/fimmu.2022.1026052

Figure Lengend Snippet: Effect of mRNA dose and nucleoside modification on immune responses. BALB/cJ mice were immunized intramuscularly (IM), two times at three-week intervals, with a low dose (10µg) or a high dose (30µg) of pfceltos mRNA that was N-glycosylation site modified (GM), containing human IgE signal sequence (IgE-SS) (GM IgE-SS), and with nucleoside ψ-pseudouridine and 5’-methylcytosine substitutions (PU5MC), (GM PU5MC IgE-SS), in LNP (LNP1 or LNP3), ( n= 5 per group). (A) Antigen-specific IgG antibody concentrations against PfCelTOS were quantified in sera two weeks after the final dose by ELISA. Antibody concentrations are represented as the mean and standard deviation (SD). Statistical analysis was performed using an unpaired t-test (*p<0.05). (B, C) IFN-γ and IL-4 cytokines were detected by ELISpot. The mean number of spot-forming cells (SFC) per splenocytes were reported with standard errors of the mean (SEM). Statistical analysis was performed using Mann-Whitney test, (*p<0.05, **p<0.01).

Article Snippet: To determine cellular responses against PfCelTOS, mouse IFN-γ and IL-4 ELISpot assay (R&D systems SEL485 SEL404, respectively) were performed according to the manufacturer’s instructions.

Techniques: Modification, Sequencing, Enzyme-linked Immunosorbent Assay, Standard Deviation, Enzyme-linked Immunospot, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Exploring in vitro expression and immune potency in mice using mRNA encoding the Plasmodium falciparum malaria antigen, CelTOS

doi: 10.3389/fimmu.2022.1026052

Figure Lengend Snippet: A three-dose regime overcomes an “all-or-none” pattern for humoral immune responses. Mice were immunized intramuscularly (IM) thrice at a three-week interval with 10µg of University of Pennsylvania (UPenn) N-glycosylation site modified (GM) and encapsulated in LNP1 (UPenn GM LNP1), or without N-glycosylation modification (UPenn LNP1) that were one-methylpseudouridine (m1Ψ)-5′-triphosphate modified, and cellulose affinity purified or with TriLink pfceltos mRNA with N-glycosylation site modified (GM) in LNP1 (TriLink GM LNP1) or LNP3 (TriLink GM LNP3) that were ψ-pseudouridine and 5-methylcytosine modified ( n = 15 per group; n = 10 in LNP alone groups). (A) Kinetics of PfCelTOS antibody concentrations measured by ELISA. Antibody concentrations are reported as the geometric mean and 95% confidence intervals. (B) IFN-γ cytokine responses were detected by ELISpot, (n=5 per group). The mean number of spot-forming cells (SFC) per splenocytes were reported with standard errors of the mean (SEM). Statistical analysis was performed using Mann-Whitney test, (*p<0.05, **p<0.01).

Article Snippet: To determine cellular responses against PfCelTOS, mouse IFN-γ and IL-4 ELISpot assay (R&D systems SEL485 SEL404, respectively) were performed according to the manufacturer’s instructions.

Techniques: Modification, Affinity Purification, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, MANN-WHITNEY