Journal: NPJ Vaccines

Article Title: A candidate subunit vaccine induces protective immunity against Mycobacterium avium subspecies paratuberculosis in mice

doi: 10.1038/s41541-023-00675-1

Figure Lengend Snippet: a Schematic diagram of the vaccination timeline. C57BL/6 mice were subcutaneously injected two times (three weeks apart) with 50 μg 66NC fusion protein formulated in Montanide ISA 61 VG adjuvant. Control groups include mice immunized with Montanide ISA 61 VG adjuvant alone as a negative control or 74 F mixed MPL adjuvant as subunit vaccine control. Three weeks after the booster immunization, the mice were bled and sacrificed. b Antigen-specific IgG1 and IgG2a isotypes were measured by ELISA. c Pictures of the IFN-γ and IL-4 ELISpot assay (representative of one experiment with at least five independent replicates). d , e Quantitation of the IFN-γ and IL-4 ELISpot assay. f The percentage of cytokine-positive (%Cyt + ) antigen-specific T-lymphocytes was quantified by intracellular cytokine staining (ICS) in CD4 + and CD8 + following stimulation of splenic T lymphocytes with the indicated antigen. g Serum cytokines levels of IFN-γ, TNF-α, and IL-17A were measured by ELISA. d – g Error bars indicate the Standard Error of Mean (SEM). One-way ANOVA was used to analyze the statistical significance, followed by Tukey’s multiple comparisons tests. ** P < 0.01, *** P < 0.001.

Article Snippet: Antibody PE/Cy5 Rat IgG1 (1:50, RG1, NBP1-43076, Novus Biologicals, Littleton, Co, USA), Alexa Fluor 647 Rat IgG1 (1:50, KLH/G1-2-2, 0116-31, SouthernBiotech, Birmingham, AL, USA), and eFluor 450 Rat IgG1 (1:50, eBRG1, 48-4301-82, ThermoFisher) were used according to the manufacturer’s instructions for isotype control.

Techniques: Injection, Adjuvant, Control, Negative Control, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Quantitation Assay, Staining

Journal: NPJ Vaccines

Article Title: A candidate subunit vaccine induces protective immunity against Mycobacterium avium subspecies paratuberculosis in mice

doi: 10.1038/s41541-023-00675-1

Figure Lengend Snippet: a Schematic diagram of the vaccination and MAP challenge timeline. C57BL/6 mice were subcutaneously injected two times (three weeks apart) with 50 μg 66NC fusion protein formulated in Montanide ISA 61 VG (61 VG) adjuvant. Control groups include mice immunized with Montanide ISA 61 VG adjuvant alone as a negative control or 74 F mixed MPL adjuvant as subunit vaccine control. Three weeks after the booster immunization, the mice were challenged with MAP K-10 by intraperitoneal injection. b Body weight was measured for 2, 4, 8, and 12 weeks after the MAP challenge ( n = 6 per group). Monitor IgG ( c ) and IgM ( d ) antibody levels. Sera were collected and measured for specific anti-66NC or 74 F antibody response of both IgG and IgM at 2, 4, 6, 8, 10, 12, 14, 16, and 18 weeks after prime vaccination by ELISA. Bacterial load quantification was evaluated in the liver ( e ) and intestine ( f ) from mice challenged by intraperitoneal injection with MAP K-10 for 2, 4, and 8 weeks after vaccination. g Representative images of Ziehl-Neelsen acid-fast stain in the liver of vaccinated mice, two weeks after being challenged with MAP, are presented (scale bars, 100 μm (top) and 10 μm (bottom)). The bottom images are enlarged from the outlined areas of the top images. b , e , f Data indicate cumulative results from at least three to six independent replicates. Mean ± SEM and Two-way ANOVA were used to analyze the statistical significance, followed by Tukey’s multiple comparisons tests. ns, non-significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Antibody PE/Cy5 Rat IgG1 (1:50, RG1, NBP1-43076, Novus Biologicals, Littleton, Co, USA), Alexa Fluor 647 Rat IgG1 (1:50, KLH/G1-2-2, 0116-31, SouthernBiotech, Birmingham, AL, USA), and eFluor 450 Rat IgG1 (1:50, eBRG1, 48-4301-82, ThermoFisher) were used according to the manufacturer’s instructions for isotype control.

Techniques: Injection, Adjuvant, Control, Negative Control, Enzyme-linked Immunosorbent Assay, Ziehl-Neelsen Stain