Journal: Pharmaceutics

Article Title: A Multivalent mRNA Therapeutic Vaccine Exhibits Breakthroughs in Immune Tolerance and Virological Suppression of HBV by Stably Presenting the Pre-S Antigen on the Cell Membrane

doi: 10.3390/pharmaceutics17020211

Figure Lengend Snippet: In vitro expression analysis of hepatitis B surface antigen mRNA vaccines. LNP-encapsulated L mRNA, M mRNA, and S mRNA were transfected into 293T cells in different combinations, and the dosage of each single mRNA was 5 μg for each treatment. ( a ) The proportion of cells expressing pre-S2 antigen or S antigen on the surface was detected by FCM at 48 h and 96 h after transfection, respectively. The results of FCM were showed using density maps. ( b ) WB analysis of the expression or secretion of the L protein (42 kDa), M protein (31 kDa), and S protein (27 kDa) in the cell membrane or supernatants at 48 h after transfection.

Article Snippet: An HBsAg ELISA Kit (E-EL-H6080, Elabscience Biotechnology Co., Ltd., Nanjing, China) was used to quantify the amount of HBsAg in the VLPs.

Techniques: In Vitro, Expressing, Vaccines, Transfection, Membrane

Journal: Pharmaceutics

Article Title: A Multivalent mRNA Therapeutic Vaccine Exhibits Breakthroughs in Immune Tolerance and Virological Suppression of HBV by Stably Presenting the Pre-S Antigen on the Cell Membrane

doi: 10.3390/pharmaceutics17020211

Figure Lengend Snippet: Humoral immune response analysis of HBV Tg mice immunized with the hepatitis B surface antigen mRNA vaccines. ( a ) Schematic diagram of the experimental protocol for the immunization of BALB/c HBV Tg mice with different combinations of mRNAs encoding hepatitis B surface antigens or with control vaccines. ( b ) CLIA detection of serum HBsAb levels in HBV Tg mice at weeks 0–32 of the immunization experiment. ( c ) Analysis of changes in the serum HBsAb concentration in mice whose HBsAb concentration was consistently less than 2000 IU/L and greater than 0 IU/L. ( d ) Correlation analysis between the serum levels of HBsAb and reduced HBsAg levels. ( e ) Indirect ELISA analysis of the binding ability of induced serum IgG to recombinant HBsAg, the pre-S peptide, or the LMS VLP after immunization with different vaccines. ( f ) Variable slope (four parameters) analysis via nonlinear regression (curve fit) was performed for the pre-S antigen-specific indirect ELISA. Representative results are presented as the means ± standard deviations (SDs).

Article Snippet: An HBsAg ELISA Kit (E-EL-H6080, Elabscience Biotechnology Co., Ltd., Nanjing, China) was used to quantify the amount of HBsAg in the VLPs.

Techniques: Vaccines, Control, Concentration Assay, Indirect ELISA, Binding Assay, Recombinant

Journal: Pharmaceutics

Article Title: A Multivalent mRNA Therapeutic Vaccine Exhibits Breakthroughs in Immune Tolerance and Virological Suppression of HBV by Stably Presenting the Pre-S Antigen on the Cell Membrane

doi: 10.3390/pharmaceutics17020211

Figure Lengend Snippet: Serological and virological response analysis of pAAV HBV1.2 mice immunized with hepatitis B surface antigen mRNA vaccines. ( a ) Schematic diagram of the experimental protocol for immunization of pAAV HBV1.2 mice with LMS mRNA vaccines, S mRNA, or GFP mRNA. Changes in serum ( b ) HBsAg, ( c ) HBsAb, and ( d ) HBeAg from 0 to 5 weeks after immunization of pAAV HBV-1.2 mice with different vaccines. ( e ) Q–PCR detection of serum HBV DNA levels in immunized mice at week 0 and week 5. Representative results are presented as the means ± SDs.

Article Snippet: An HBsAg ELISA Kit (E-EL-H6080, Elabscience Biotechnology Co., Ltd., Nanjing, China) was used to quantify the amount of HBsAg in the VLPs.

Techniques: Vaccines

Journal: Pharmaceutics

Article Title: A Multivalent mRNA Therapeutic Vaccine Exhibits Breakthroughs in Immune Tolerance and Virological Suppression of HBV by Stably Presenting the Pre-S Antigen on the Cell Membrane

doi: 10.3390/pharmaceutics17020211

Figure Lengend Snippet: Serological and virological response analysis of rAAV8 HBV1.3 mice immunized with hepatitis B surface antigen mRNA vaccines. ( a ) Schematic diagram of the experimental protocol for immunization of rAAV8 HBV1.3 (1 × 10 10 vg/mouse) mice with hepatitis B surface antigen mRNA vaccines in different combinations, S mRNA, or GFP mRNA. Changes in serum ( b ) HBsAg, ( c ) HBsAb, and ( d ) HBeAg from 0 to 9 weeks of immunization with different vaccines. ( e ) Q–PCR detection of serum HBV DNA levels in immunized mice at week 0 and week 9. Representative results are presented as the means ± SDs.

Article Snippet: An HBsAg ELISA Kit (E-EL-H6080, Elabscience Biotechnology Co., Ltd., Nanjing, China) was used to quantify the amount of HBsAg in the VLPs.

Techniques: Vaccines

Journal: Pharmaceutics

Article Title: A Multivalent mRNA Therapeutic Vaccine Exhibits Breakthroughs in Immune Tolerance and Virological Suppression of HBV by Stably Presenting the Pre-S Antigen on the Cell Membrane

doi: 10.3390/pharmaceutics17020211

Figure Lengend Snippet: The analysis of antigen-dependent T-cell responses activated by Hepatitis B surface antigen mRNA vaccines. ( a ) BALB/3T3 cells were transiently transfected with pD2531.L, pD2531.M, and pD2531.S plasmids (1:1:1 mass ratio) via Lipofectamine 3000, and FCM was performed to detect pre-S2 antigen and S antigen expression on the cell surface. ( b ) After immunization of BALB/c mice with different hepatitis B surface antigen mRNA vaccines or PBS, splenic CD4 + T cells were isolated and stimulated with BALB/3T3 cells transiently expressing the LMS antigen. Meanwhile, antigen-incubated T cells from PBS-immunized mice were used as negative control and CD3/CD28 bead-stimulated T cells from PBS-immunized mice were used as positive control. FCM was used to measure the level of IFN-γ in CD4 + T cells after the addition of brefeldin A.

Article Snippet: An HBsAg ELISA Kit (E-EL-H6080, Elabscience Biotechnology Co., Ltd., Nanjing, China) was used to quantify the amount of HBsAg in the VLPs.

Techniques: Vaccines, Transfection, Expressing, Isolation, Incubation, Negative Control, Positive Control

Journal: Pharmaceutics

Article Title: A Multivalent mRNA Therapeutic Vaccine Exhibits Breakthroughs in Immune Tolerance and Virological Suppression of HBV by Stably Presenting the Pre-S Antigen on the Cell Membrane

doi: 10.3390/pharmaceutics17020211

Figure Lengend Snippet: Schematic diagram of the mechanism by which the LMS mRNA therapeutic vaccine disrupts HBsAg-mediated immune tolerance and reactivates immune responses against HBV. The numbers 1–6 marked on the membrane structure of L protein represent intracellular structure of S antigen, T cell recognition epitopes of pre-S1 antigen, hepatocyte surface receptor NTCP binding site for pre-S1 antigen, S antigen (highly variable region) with a-antigen determinant, lipid membrane, hepatocyte binding region for pre-S2 antigen, membrane attachment region of HBV-infected hepatocytes, respectively.

Article Snippet: An HBsAg ELISA Kit (E-EL-H6080, Elabscience Biotechnology Co., Ltd., Nanjing, China) was used to quantify the amount of HBsAg in the VLPs.

Techniques: Membrane, Binding Assay, Infection