Journal: Scientific Reports
Article Title: Codon optimization and improved delivery/immunization regimen enhance the immune response against wild-type and drug-resistant HIV-1 reverse transcriptase, preserving its Th2-polarity
Figure Lengend Snippet: Immune responses following immunization with expression-optimized RT genes. BALB/c mice (n = 5 or 6) were immunized with two intradermal injections (29G needle) containing 20 µg of RTwt, RTwt-opt, RTwt-opt-in, RT1.14, RT1.14-opt, or RT1.14-opt-in encoding plasmids per mouse with subsequent electroporation by Dermavax (standard protocol) in two independent immunizations. At 21 days post-immunization mice were sacrificed, and sera and splenocytes were isolated for further immune tests. End-point average titers of anti-RT total IgG and IgG subtypes were detected using ELISA against recombinant RTwt and RT1.14 proteins with cut-offs set against the serum reactivity of control mice immunized with vector pVax 1. ( a – c ) ELISA with the identical RT protein variant received as a gene, i.e. recombinant RTwt protein for RTwt, RTwt-opt, and RTwt-opt-in immunized mice or RT1.14 for RT1.14, RT1.14-opt, and RT1.14-opt-in immunized mice. ( a ) ELISA with the homologous RT, i.e. recombinant RT1.14 protein for RTwt, RTwt-opt, and RTwt-opt-in or RTwt protein for RT1.14, RT1.14-opt, and RT1.14-opt-in immunized mice. ( b ) IgG2a/IgG1 ratio for antibody reactivity against RT variants matching RT used as DNA immunogen ( c ). In panels (a,b) the asterisk designates the difference between RTwt and RTwt-opt/RTwt-opt-in and between RT1.14 and RT1.14-opt/RT1.14-opt-in variants for all IgG subtypes. Murine splenocytes were stimulated in vitro with an RT-derived peptide representing the epitope of RT aa 528–543 (immunodominant T-cell epitope; Table 2 ) in the IFN-γ/IL-2 Fluorospot test ( d – f ). The average number of cells was registered as signal-forming units (sfu) per million splenocytes secreting IFN-γ ( d ) IL-2 ( e ) and IFN-γ/IL-2 ( f ) and the error bars represent the SD. *p
Article Snippet: In our previous studies, we employed Dermavax EP (Cellectis) and MN electrodes .
Techniques: Expressing, Mouse Assay, Electroporation, Isolation, Enzyme-linked Immunosorbent Assay, Recombinant, Plasmid Preparation, Variant Assay, In Vitro, Derivative Assay